Martin Scanlon

Controlled Construction of Cyclic d / l Peptide Nanorods.

Publisher: Wiley

Date: 05-12-2019

DOI: 10.1002/ANIE.201811910

Abstract: Cyclic d / l peptides (CPs) assemble spontaneously via backbone H-bonding to form extended nanostructures. These modular materials have great potential as versatile bionanomaterials. However, the useful development of CP nanomaterials requires practical methods to direct and control their assembly. In this work, we present novel, heterogeneous, covalently linked CP tetramers that achieve local control over the CP subunit order and composition through coupling of amino acid side-chains using copper-activated azide-alkyne cycloaddition and disulfide bond formation. Cryo-transmission electron microscopy revealed the formation of highly ordered, fibrous nanostructures, while NMR studies showed that these systems have strong intramolecular H-bonding in solution. The introduction of inter-CP tethers is expected to enable the development of complex nanomaterials with controllable chemical properties, facilitating the development of precisely functionalized or "decorated" peptide nanostructures.

The Dotted Cap Notation: A concise notation for describing variegated dendrimers

Publisher: Royal Society of Chemistry (RSC)

Date: 2008

DOI: 10.1039/B800724A

Substituted 1-methyl-4-phenylpyrrolidin-2-ones – Fragment-based design of N-methylpyrrolidone-derived bromodomain inhibitors

Publisher: Elsevier BV

Date: 04-2020

DOI: 10.1016/J.EJMECH.2020.112120

Difficult Macrocyclizations:  New Strategies for Synthesizing Highly Strained Cyclic Tetrapeptides

Publisher: American Chemical Society (ACS)

Date: 07-2003

DOI: 10.1021/OL034907O

Abstract: [reaction: see text] Cyclic tetrapeptides are an intriguing class of natural products. To synthesize highly strained cyclic tetrapeptides we developed a macrocyclization strategy that involves the inclusion of 2-hydroxy-6-nitrobenzyl (HnB) group at the N-terminus and in the "middle" of the sequence. The N-terminal auxiliary performs a ring closure/ring contraction role, and the backbone auxiliary promotes cis amide bonds to facilitate the otherwise difficult ring contraction. Following this route, the all-L cyclic tetrapeptide cyclo-[Tyr-Arg-Phe-Ala] was successfully prepared.

A Marine Snail Neurotoxin Shares with Scorpion Toxins a Convergent Mechanism of Blockade on the Pore of Voltage-Gated K Channels

Publisher: Rockefeller University Press

Date: 07-1999

DOI: 10.1085/JGP.114.1.141

Abstract: κ-Conotoxin-PVIIA (κ-PVIIA) belongs to a family of peptides derived from a hunting marine snail that targets to a wide variety of ion channels and receptors. κ-PVIIA is a small, structurally constrained, 27-residue peptide that inhibits voltage-gated K channels. Three disulfide bonds shape a characteristic four-loop folding. The spatial localization of positively charged residues in κ-PVIIA exhibits strong structural mimicry to that of charybdotoxin, a scorpion toxin that occludes the pore of K channels. We studied the mechanism by which this peptide inhibits Shaker K channels expressed in Xenopus oocytes with the N-type inactivation removed. Chronically applied to whole oocytes or outside-out patches, κ-PVIIA inhibition appears as a voltage-dependent relaxation in response to the depolarizing pulse used to activate the channels. At any applied voltage, the relaxation rate depended linearly on the toxin concentration, indicating a bimolecular stoichiometry. Time constants and voltage dependence of the current relaxation produced by chronic applications agreed with that of rapid applications to open channels. Effective valence of the voltage dependence, zδ, is ∼0.55 and resides primarily in the rate of dissociation from the channel, while the association rate is voltage independent with a magnitude of 107–108 M−1 s−1, consistent with diffusion-limited binding. Compatible with a purely competitive interaction for a site in the external vestibule, tetraethylammonium, a well-known K-pore blocker, reduced κ-PVIIA's association rate only. Removal of internal K+ reduced, but did not eliminate, the effective valence of the toxin dissociation rate to a value & .3. This trans-pore effect suggests that: (a) as in the α-KTx, a positively charged side chain, possibly a Lys, interacts electrostatically with ions residing inside the Shaker pore, and (b) a part of the toxin occupies an externally accessible K+ binding site, decreasing the degree of pore occupancy by permeant ions. We conclude that, although evolutionarily distant to scorpion toxins, κ-PVIIA shares with them a remarkably similar mechanism of inhibition of K channels.

Selective Binding of Small Molecules to Vibrio cholerae DsbA Offers a Starting Point for the Design of Novel Antibacterials.

Publisher: Wiley

Date: 27-01-2022

DOI: 10.1002/CMDC.202100673

Abstract: DsbA enzymes catalyze oxidative folding of proteins that are secreted into the periplasm of Gram‐negative bacteria, and they are indispensable for the virulence of human pathogens such as Vibrio cholerae and Escherichia coli . Therefore, targeting DsbA represents an attractive approach to control bacterial virulence. X‐ray crystal structures reveal that DsbA enzymes share a similar fold, however, the hydrophobic groove adjacent to the active site, which is implicated in substrate binding, is shorter and flatter in the structure of V. cholerae DsbA (VcDsbA) compared to E. coli DsbA (EcDsbA). The flat and largely featureless nature of this hydrophobic groove is challenging for the development of small molecule inhibitors. Using fragment‐based screening approaches, we have identified a novel small molecule, based on the benzimidazole scaffold, that binds to the hydrophobic groove of oxidized VcDsbA with a K D of 446±10 μM. The same benzimidazole compound has ∼8‐fold selectivity for VcDsbA over EcDsbA and binds to oxidized EcDsbA, with K D .5 mM. We generated a model of the benzimidazole complex with VcDsbA using NMR data but were unable to determine the structure of the benzimidazole bound EcDsbA using either NMR or X‐ray crystallography. Therefore, a structural basis for the observed selectivity is unclear. To better understand ligand binding to these two enzymes we crystallized each of them in complex with a known ligand, the bile salt sodium taurocholate. The crystal structures show that taurocholate adopts different binding poses in complex with VcDsbA and EcDsbA, and reveal the protein‐ligand interactions that stabilize the different modes of binding. This work highlights the capacity of fragment‐based drug discovery to identify inhibitors of challenging protein targets. In addition, it provides a starting point for development of more potent and specific VcDsbA inhibitors that act through a novel anti‐virulence mechanism.

Molecular Insights into the Interaction Between the SPRY Domain-Containing SOCS Box Protein SPSB2 and Peptides Based on the Binding Motif from iNOS

Publisher: CSIRO Publishing

Date: 2017

DOI: 10.1071/CH16510

Abstract: SPRY domain-containing SOCS box proteins SPSB1, 2, and 4 mediate the proteasomal degradation of inducible nitric oxide synthase (iNOS) and thereby modulate the amount of NO available for combating infectious organisms. A highly conserved Asp-Ile-Asn-Asn-Asn (DINNN) motif found at the N-terminus of iNOS binds to SPSB2 with nanomolar affinity. The design of specific and potent inhibitors of iNOS–SPSB interactions will be aided by a better understanding of the interactions of this DINNN sequence with SPSB2. Although crystal structures of SPSB complexes with DINNN peptides are available, aspects of the interaction between peptide and protein are still not fully understood. Here, our results from surface plasmon resonance and NMR spectroscopy indicate that residues flanking the DINNN motif, which make no direct contact with SPSB2 in the available crystal structures, nonetheless play an important role in enhancing the binding affinity to SPSB2, by up to 80-fold. Mutational analysis of the DINNN sequence showed that mutation of the Asp or the first Asn residue to Ala reduced the binding affinity by 200- or 600-fold respectively, whereas mutation of the third Asn made binding undetectable. Ala substitution of the second Asn residue caused a 30-fold drop in binding affinity. Substitution of the Ile had very little effect on the binding affinity and substitutions with bulky residues were tolerated. This provides an opportunity for further modification for therapeutic applications. These results highlight the complex interplay of peptide sequence and protein binding and inform efforts to design peptide therapeutics to disrupt the iNOS–SPSB interaction.

Propargyloxyproline Regio- and Stereoisomers for Click-Conjugation of Peptides: Synthesis and Application in Linear and Cyclic Peptides

Publisher: CSIRO Publishing

Date: 2015

DOI: 10.1071/CH15146

Abstract: The use of the click reaction for the introduction of conjugate groups, such as affinity or fluorescent labels, to a peptide for the study of peptide biochemistry and pharmacology is widespread. However, the nature and location of substituted 1,2,3-triazoles in peptide sequences may markedly affect conformation or binding as compared with native sequences. We have examined the preparation and application of propargyloxyproline (Pop) residues as a precursor to such peptide conjugates. Pop residues are available in a range of regio- and stereoisomers from hydroxyproline precursors and are readily prepared in Fmoc-protected form. They can be incorporated routinely in peptide synthesis and broadly retain the conformational properties of the parent proline containing peptides. This is exemplified by the preparation of biotin- and fluorophore-labelled peptides derived from linear and cyclic peptides.

Design of a Fragment-Screening Library

Publisher: Elsevier

Date: 2018

DOI: 10.1016/BS.MIE.2018.09.018

Abstract: Herein we describe a method for the design, purchase, and assembly of a fragment-screening library from a list of commercially available compounds. The computational tools used in assessment of compound properties as well as the workflow for compound selection are provided for reference as implemented in commercially available software that is free and accessible to most academic users. The workflow can be modified as necessary to generate a fit-for-purpose fragment library with the desired compound property profiles. An analytical process for assessing the quality, identity, and suitability of a purchased fragment for inclusion in a screening collection is described. Results from our in-house library are presented as an ex le of compound progression through this quality control process.

Primary sequence determination and molecular modelling of the variable region of an antiMUC1 mucin monoclonal antibody

Publisher: Elsevier BV

Date: 1995

DOI: 10.1016/0959-8049(94)00431-4

Abstract: Polymerase chain reaction (PCR) products representative of the DNA sequence coding for the variable heavy (VH) and the variable light (VL) chains of an antiMUC1 mucin monoclonal antibody, C595, have been produced. These products were cloned, sequenced, and the primary amino acid sequences of the VH and VL regions deduced. The hypervariable complementarity determining regions (CDRs) and framework regions in the heavy and light chains were located, and homologies with canonical forms for the CDR loops L1, L2, L3, H1 and H2 were identified by database searching. The structure for the H3 loop was calculated directly. Computational molecular modelling was accomplished using the fully automated AbM package (Oxford Molecular, Oxford, U.K.). Energy minimisation was performed using the program InsightII (Biosym, San Diego, California, U.S.A.). The investigation provides a basis for the molecular analysis of the antigen binding site of the C595 antibody with the aim to identify key residues and interactions involved in the immune recognition of the C595 antibody defined epitope, which is expressed in the majority of breast and ovarian carcinomas.

Characterization of the Drug Binding Specificity of Rat Liver Fatty Acid Binding Protein

Publisher: American Chemical Society (ACS)

Date: 07-2008

DOI: 10.1021/JM701192W

Abstract: Liver-fatty acid binding protein (L-FABP) is found in high levels in enterocytes and is involved in the cytosolic solubilization of fatty acids during fat absorption. In the current studies, the interaction of L-FABP with a range of lipophilic drugs has been evaluated to explore the potential for L-FABP to provide an analogous function during the absorption of lipophilic drugs. Binding affinity for L-FABP was assessed by displacement of a fluorescent marker, 1-anilinonaphthalene-8-sulfonic acid (ANS), and the binding site location was determined via nuclear magnetic resonance chemical shift perturbation studies. It was found that the majority of drugs bound to L-FABP at two sites, with the internal site generally having a higher affinity for the compounds tested. Furthermore, in contrast to the interaction of L-FABP with fatty acids, it was demonstrated that a terminal carboxylate is not required for specific binding of lipophilic drugs at the internal site of L-FABP.

Backbone assignments of the 34 kDa ketopantoate reductase from E. coli

Publisher: Springer Science and Business Media LLC

Date: 26-04-2008

DOI: 10.1007/S12104-008-9093-9

Abstract: Ketopantoate reductase is an essential enzyme for pantothenate (vitamin B5) synthesis and a potential antibiotic target. Here we report the 15N and 1HN, 13C', 13C(alpha) and 13C(beta) chemical shift assignments of the 34 kDa ketopantoate reductase in its apo state.

Multiple conformations of the sea anemone polypeptide anthopleurin‐A in solution

Publisher: Wiley

Date: 07-1994

DOI: 10.1002/PRO.5560030717

Abstract: Anthopleurin-A (AP-A) is a member of a family of sea anemone-derived polypeptides that interact with sodium channels in a voltage-dependent manner, producing a positive inotropic effect on the mammalian heart. There has been considerable interest in this molecule as a lead compound for the development of novel therapeutic agents. Earlier attempts to define the 3-dimensional structure of AP-A were complicated by the fact that it was found to exist in 2 conformations in solution. Using 1H- and 13C-NMR spectroscopy, we have now shown that this conformational heterogeneity arises from cis-trans isomerization about the Gly 40-Pro 41 peptide bond and that in the major form of the protein this peptide bond adopts a cis conformation. Furthermore, the increased sensitivity afforded by higher-field NMR has allowed identification of additional minor conformations of AP-A, the origin of which is presently unknown. We believe there will be many more ex les of the detection by high-field NMR of previously unobserved minor conformations of proteins in solution.

Colloidal characteristics and formulation of pure protein particulate vaccines

Publisher: Oxford University Press (OUP)

Date: 29-05-2012

DOI: 10.1111/J.2042-7158.2012.01513.X

Abstract: We recently reported that dense gas processing of the protein ovalbumin (OVA) resulted in the formation of particles that were insoluble in water and which retained their immunogenicity in vivo. In the present study, the colloidal properties of these pure protein particles were investigated to in part inform rational formulation approaches. The colloidal properties of the particles, in terms of size, zeta potential and pH-dependent surface and solution properties, were examined. In phosphate-buffered saline (pH 7.4), flocculation of the particles was observed, which was prevented when particles were suspended in acetate buffer at pH lower than 4. The resulting particle size was 300 nm with low polydispersity and zeta potential of 22.9 ± 3.1 mV (mean ± SEM, n = 3) at pH 3. Dense gas OVA particles were also prevented from flocculation using steric stabilisation with Pluronic F127. In this form the particles were stable in Krebs–Henseleit solution for 48 h at room temperature. These findings indicate that insoluble pure protein particles produced by dense gas processing have desirable characteristics as particulate vaccines, including consistency of particle size under controlled conditions and high colloid stability.

Parallel Screening of Low Molecular Weight Fragment Libraries: Do Differences in Methodology Affect Hit Identification?

Publisher: Elsevier BV

Date: 02-2013

DOI: 10.1177/1087057112465979

Abstract: Fragment screening is becoming widely accepted as a technique to identify hit compounds for the development of novel lead compounds. In neighboring laboratories, we have recently, and independently, performed a fragment screening c aign on the HIV-1 integrase core domain (IN) using similar commercially purchased fragment libraries. The two c aigns used different screening methods for the preliminary identification of fragment hits one used saturation transfer difference nuclear magnetic resonance spectroscopy (STD-NMR), and the other used surface plasmon resonance (SPR) spectroscopy. Both initial screens were followed by X-ray crystallography. Using the STD-NMR/X-ray approach, 15 IN/fragment complexes were identified, whereas the SPR/X-ray approach found 6 complexes. In this article, we compare the approaches that were taken by each group and the results obtained, and we look at what factors could potentially influence the final results. We find that despite using different approaches with little overlap of initial hits, both approaches identified binding sites on IN that provided a basis for fragment-based lead discovery and further lead development. Comparison of hits identified in the two studies highlights a key role for both the conditions under which fragment binding is measured and the criteria selected to classify hits.

Redox‐stable cyclic peptide inhibitors of the SPSB2–iNOS interaction

Publisher: Wiley

Date: 03-2016

DOI: 10.1002/1873-3468.12115

Abstract: SPSB2 mediates the proteasomal degradation of iNOS. Inhibitors of SPSB2-iNOS interaction are expected to prolong iNOS lifetime and thereby enhance killing of persistent pathogens. Here, we describe the synthesis and characterization of two redox-stable cyclized peptides containing the DINNN motif required for SPSB2 binding. Both analogues bind with low nanomolar affinity to the iNOS binding site on SPSB, as determined by SPR and (19)F NMR, and efficiently displace full-length iNOS from binding to SPSB2 in macrophage cell lysates. These peptides provide a foundation for future development of redox-stable, potent ligands for SPSB proteins as a potential novel class of anti-infectives.

Cyclooligomerization of Thiazole-Containing Tetrapeptides. Symmetrical Macrocycles with up to 76 Amino Acids

Publisher: American Chemical Society (ACS)

Date: 03-1999

DOI: 10.1021/JA983354N

The Three-dimensional Solution Structure of NaD1, a New Floral Defensin from Nicotiana alata and its Application to a Homology Model of the Crop Defense Protein alfAFP

Publisher: Elsevier BV

Date: 2003

DOI: 10.1016/S0022-2836(02)01103-8

Abstract: NMR spectroscopy and simulated annealing calculations have been used to determine the three-dimensional structure of NaD1, a novel antifungal and insecticidal protein isolated from the flowers of Nicotiana alata. NaD1 is a basic, cysteine-rich protein of 47 residues and is the first ex le of a plant defensin from flowers to be characterized structurally. Its three-dimensional structure consists of an alpha-helix and a triple-stranded antiparallel beta-sheet that are stabilized by four intramolecular disulfide bonds. NaD1 features all the characteristics of the cysteine-stabilized alphabeta motif that has been described for a variety of proteins of differing functions ranging from antibacterial insect defensins and ion channel-perturbing scorpion toxins to an elicitor of the sweet taste response. The protein is biologically active against insect pests, which makes it a potential candidate for use in crop protection. NaD1 shares 31% sequence identity with alfAFP, an antifungal protein from alfalfa that confers resistance to a fungal pathogen in transgenic potatoes. The structure of NaD1 was used to obtain a homology model of alfAFP, since NaD1 has the highest level of sequence identity with alfAFP of any structurally characterized antifungal defensin. The structures of NaD1 and alfAFP were used in conjunction with structure-activity data for the radish defensin Rs-AFP2 to provide an insight into structure-function relationships. In particular, a putative effector site was identified in the structure of NaD1 and in the corresponding homology model of alfAFP.

Backbone and side chain H-1, N-15 and C-13 assignments for the oxidised and reduced forms of the oxidoreductase protein DsbA from Staphylococcus aureus

Publisher: Springer Science and Business Media LLC

Date: 20-11-2010

DOI: 10.1007/S12104-009-9199-8

Abstract: The function and dynamics of the thiol-disulfide oxidoreductase DsbA in the low-GC gram positive bacterium, Staphylococcus aureus, are yet to be elucidated. Here we report 13C, 15N and 1H assignments for the oxidised and reduced forms of SaDsbA as a prelude to further studies on the enzyme.

The Structure of the Bacterial Oxidoreductase Enzyme DsbA in Complex with a Peptide Reveals a Basis for Substrate Specificity in the Catalytic Cycle of DsbA Enzymes

Publisher: Elsevier BV

Date: 06-2009

DOI: 10.1074/JBC.M109.011502

Probing the Fibrate Binding Specificity of Rat Liver Fatty Acid Binding Protein

Publisher: American Chemical Society (ACS)

Date: 07-08-2009

DOI: 10.1021/JM801349E

Abstract: Liver-fatty acid binding protein (L-FABP) is found in high levels in enterocytes and is involved in cytosolic solubilization of fatty acids. In addition, L-FABP has been shown to bind endogenous and exogenous lipophilic compounds, suggesting that it may also play a role in modulating their absorption and disposition within enterocytes. Previously, we have described binding of L-FABP to a range of drugs, including a series of fibrates. In the present study, we have generated structural models of L-FABP-fibrate complexes and undertaken thermodynamic analysis of the binding of fibrates containing either a carboxylic acid or ester functionality. Analysis of the current data reveals that both the location and the energetics of binding are different for fibrates that contain a carboxylate compared to those that do not. As such, the data presented in this study suggest potential mechanisms that underpin molecular recognition and dictate specificity in the interaction between fibrates and L-FABP.

Rapid Elaboration of Fragments into Leads by X-ray Crystallographic Screening of Parallel Chemical Libraries (REFiL_X)

Publisher: American Chemical Society (ACS)

Date: 12-06-2020

DOI: 10.1021/ACS.JMEDCHEM.0C00111

Solution structure and DNA binding of the catalytic domain of the large serine resolvase TnpX

Publisher: Wiley

Date: 26-02-2015

DOI: 10.1002/JMR.2446

Abstract: The transfer of antibiotic resistance between bacteria is mediated by mobile genetic elements such as plasmids and transposons. TnpX is a member of the large serine recombinase subgroup of site-specific recombinases and is responsible for the excision and insertion of mobile genetic elements that encode chlor henicol resistance in the pathogens Clostridium perfringens and Clostridium difficile. TnpX consists of three structural domains: domain I contains the catalytic site, whereas domains II and III contain DNA-binding motifs. We have solved the solution structure of residues 1-120 of the catalytic domain I of TnpX. The TnpX catalytic domain shares the same overall fold as other serine recombinases however, differences are evident in the identity of the proposed hydrogen donor and in the size, amino acid composition, conformation, and dynamics of the TnpX active site loops. To obtain the interaction surface of TnpX1-120 , we titrated a DNA oligonucleotide containing the circular intermediate joint attCI recombination site into (15) N-labeled TnpX1-120 and observed progressive nuclear magnetic resonance chemical shift perturbations using (15) N HSQC spectra. Perturbations were largely confined to a region surrounding the catalytic serine and encompassed residues of the active site loops. Utilizing the perturbation map and the data-driven docking program, HADDOCK, we have generated a model of the DNA interaction complex for the TnpX catalytic domain.

Binding of a pyrimidine RNA base-mimic to SARS-CoV-2 nonstructural protein 9

Publisher: Elsevier BV

Date: 09-2021

DOI: 10.1016/J.JBC.2021.101018

Design, Synthesis, and Characterization of Cyclic Peptidomimetics of the Inducible Nitric Oxide Synthase Binding Epitope That Disrupt the Protein–Protein Interaction Involving SPRY Domain-Containing S

Publisher: American Chemical Society (ACS)

Date: 08-06-2016

DOI: 10.1021/ACS.JMEDCHEM.6B00386

Abstract: SPRY domain-containing suppressor of cytokine signaling box protein (SPSB) 2-deficient macrophages have been found to exhibit prolonged expression of inducible nitric oxide synthase (iNOS) and enhanced killing of persistent pathogens, suggesting that inhibitors of the SPSB2-iNOS interaction have potential as novel anti-infectives. In this study, we describe the design, synthesis, and characterization of cyclic peptidomimetic inhibitors of the SPSB2-iNOS interaction constrained by organic linkers to improve stability and druggability. SPR, ITC, and (19)F NMR analyses revealed that the most potent cyclic peptidomimetic bound to the iNOS binding site of SPSB2 with low nanomolar affinity (KD 29 nM), a 10-fold improvement over that of the linear peptide DINNN (KD 318 nM), and showed strong inhibition of SPSB2-iNOS interaction in macrophage cell lysates. This study exemplifies a novel approach to cyclize a Type II β-turn linear peptide and provides a foundation for future development of this group of inhibitors as new anti-infectives.

Development of Inhibitors of Plasmodium falciparum Apical Membrane Antigen 1 Based on Fragment Screening

Publisher: CSIRO Publishing

Date: 2013

DOI: 10.1071/CH13266

Abstract: Apical membrane antigen 1 (AMA1) is an essential component of the moving junction complex used by Plasmodium falciparum to invade human red blood cells. AMA1 has a conserved hydrophobic cleft that is the site of key interactions with the rhoptry neck protein complex. Our goal is to develop small molecule inhibitors of AMA1 with broad strain specificity, which we are pursuing using a fragment-based approach. In our screening c aign, we identified fragments that bind to the hydrophobic cleft with a hit rate of 5 %. The high hit rate observed strongly suggests that a druggable pocket is present within the cleft.

The Cysteine-rich Secretory Protein Domain of Tpx-1 Is Related to Ion Channel Toxins and Regulates Ryanodine Receptor Ca2+ Signaling

Publisher: Elsevier BV

Date: 02-2006

DOI: 10.1074/JBC.M506849200

Fatty Acid–Binding Protein 5 Mediates the Uptake of Fatty Acids, but not Drugs, Into Human Brain Endothelial Cells

Publisher: Elsevier BV

Date: 04-2018

DOI: 10.1016/J.XPHS.2017.11.024

Abstract: The purpose of this study was to examine the involvement of fatty acid-binding protein 5 (FABP5), a lipid-binding protein expressed at the blood-brain barrier (BBB), in fatty acid and drug uptake into human brain endothelial cells. Following transfection with siRNA against hFABP5, human brain endothelial cell (hCMEC/D3) uptake of lipophilic ligands with varying affinity to FABP5 was assessed with intracellular concentrations quantified by liquid scintillation counting, HPLC, or LCMS/MS. The in situ BBB transport of [

Structural and Biochemical Characterization of the Oxidoreductase NmDsbA3 from Neisseria meningitidis

Publisher: Elsevier BV

Date: 11-2008

DOI: 10.1074/JBC.M803990200

Enhancing the immunogenicity and modulating the fine epitope recognition of antisera to a helical group A streptococcal peptide vaccine candidate from the M protein using lipid‐core peptide technology

Publisher: Wiley

Date: 04-2002

DOI: 10.1046/J.1440-1711.2002.01067.X

Abstract: A conserved helical peptide vaccine candidate from the M protein of group A streptococci, p145, has been described. Minimal epitopes within p145 have been defined and an epitope recognized by protective antibodies, but not by autoreactive T cells, has been identified. When administered to mice, p145 has low immunogenicity. Many boosts of peptide are required to achieve a high antibody titre (> 12 800). To attempt to overcome this low immunogenicity, lipid-core peptide technology was employed. Lipid-core peptides (LCP) consist of an oligomeric polylysine core, with multiple copies of the peptide of choice, conjugated to a series of lipoamino acids, which acts as an anchor for the antigen. Seven different LCP constructs based on the p145 peptide sequence were synthesized (LCP1-->LCP7) and the immunogenicity of the compounds examined. The most immunogenic constructs contained the longest alkyl side-chains. The number of lipoamino acids in the constructs affected the immunogenicity and spacing between the alkyl side-chains increased immunogenicity. An increase in immunogenicity (enzyme-linked immunosorbent assay (ELISA) titres) of up to 100-fold was demonstrated using this technology and some constructs without adjuvant were more immunogenic than p145 administered with complete Freund's adjuvant (CFA). The fine specificity of the induced antibody response differed for the different constructs but one construct, LCP4, induced antibodies of identical fine specificity to those found in endemic human serum. Opsonic activity of LCP4 antisera was more than double that of p145 antisera. These data show the potential for LCP technology to both enhance immunogenicity of complex peptides and to focus the immune response towards or away from critical epitopes.

Characterization of lipophilic drug binding to rat intestinal fatty acid binding protein

Publisher: Springer Science and Business Media LLC

Date: 22-01-2009

DOI: 10.1007/S11010-008-0009-X

Abstract: Intestinal fatty acid binding protein (I-FABP) is present at high levels in the absorptive cells of the intestine (enterocytes) where it plays a role in the intracellular solubilization of fatty acids (FA). However, I-FABP has also been shown to bind to a range of non-FA ligands, including some lipophilic drug molecules, albeit with generally lower affinity than FA. The significance of these lower affinity interactions with exogenous compounds is not known. In this manuscript, we describe further characterization of drug-rat I-FABP binding interactions using a thermal-shift assay. A structural explanation of the observed affinity of rat I-FABP for different drugs based on spectroscopic data and modeling experiments is presented. In addition, immunocytochemistry has been used to probe the expression of I-FABP in a cell culture model reflective of the absorptive cells of the small intestine. Taken together, these data suggest a possible role for I-FABP in the disposition of some lipophilic drugs within the enterocyte.

The 1.2 A resolution crystal structure of TcpG, the Vibrio cholerae DsbA disulfide-forming protein required for pilus and cholera-toxin production

Publisher: International Union of Crystallography (IUCr)

Date: 13-09-2012

DOI: 10.1107/S0907444912026388

Structure of the chicken CD3εδ/γ heterodimer and its assembly with the αβT cell receptor

Publisher: Elsevier BV

Date: 03-2014

DOI: 10.1074/JBC.M113.544965

The Structure of Integrin α1I Domain in Complex with a Collagen-mimetic Peptide

Publisher: Elsevier BV

Date: 12-2013

DOI: 10.1074/JBC.M113.480251

Dietary docosahexaenoic acid supplementation enhances expression of fatty acid-binding protein 5 at the blood–brain barrier and brain docosahexaenoic acid levels

Publisher: Wiley

Date: 07-2018

DOI: 10.1111/JNC.14342

Abstract: The cytoplasmic trafficking of docosahexaenoic acid (DHA), a cognitively beneficial fatty acid, across the blood-brain barrier (BBB) is governed by fatty acid-binding protein 5 (FABP5). Lower levels of brain DHA have been observed in Alzheimer's disease (AD), which is associated with diminished BBB expression of FABP5. Therefore, up-regulating FABP5 expression at the BBB may be a novel approach for enhancing BBB transport of DHA in AD. DHA supplementation has been shown to be beneficial in various mouse models of AD, and therefore, the aim of this study was to determine whether DHA has the potential to up-regulate the BBB expression of FABP5, thereby enhancing its own uptake into the brain. Treating human brain microvascular brain endothelial (hCMEC/D3) cells with the maximum tolerable concentration of DHA (12.5 μM) for 72 h resulted in a 1.4-fold increase in FABP5 protein expression. Associated with this was increased expression of fatty acid transport proteins 1 and 4. To study the impact of dietary DHA supplementation, 6- to 8-week-old C57BL/6 mice were fed with a control diet or a DHA-enriched diet for 21 days. Brain microvascular FABP5 protein expression was up-regulated 1.7-fold in mice fed the DHA-enriched diet, and this was associated with increased brain DHA levels (1.3-fold). Despite an increase in brain DHA levels, reduced BBB transport of

Peptide epitope binding and fluorescence quenching with the anti-mucin antibody C595

Publisher: Elsevier BV

Date: 30-04-1992

DOI: 10.1016/0304-3835(92)90261-S

Abstract: The Fab fragment of the anti-polymorphic epithelial mucin antibody C595 (IgG3, kappa) has been produced by digestion with papain. The purified antibody fragment retained its binding activity for the mucin in an indirect radioisotopic antiglobulin assay. The binding constant of the Fab fragment for the synthetic peptide (PDTRPAPGSTAPPAHGVTSA) corresponding to the 20-amino acid tandem repeat sequence found in the mucin protein core was determined to be 0.3 x 10(6) M-1 by measuring the capacity of the peptide to quench the fluorescence of the Fab fragment.

The ways and means of fragment-based drug design

Publisher: Elsevier BV

Date: 11-2016

DOI: 10.1016/J.PHARMTHERA.2016.07.003

Abstract: Fragment-based drug design (FBDD) has emerged as a mainstream approach for the rapid and efficient identification of building blocks that can be used to develop high-affinity ligands against protein targets. One of the strengths of FBDD is the relative ease and low cost of the primary screen to identify fragments that bind. However, the fragments that emerge from primary screens often have low affinities, with K

Fatty acid binding proteins shape the cellular response to activation of the glucocorticoid receptor

Publisher: Cold Spring Harbor Laboratory

Date: 04-07-2021

DOI: 10.1101/2021.07.02.450968

Abstract: Glucocorticoids are steroid hormones that are essential for life in mammals. Therapeutically, they are some of the most cost-effective drugs for the treatment of inflammatory diseases ranging from skin rashes to COVID-19, but their use is limited by adverse effects. Glucocorticoids exert their effects via the glucocorticoid receptor, a type I nuclear hormone receptor which modulates gene expression. The transcriptional activity of some related, but nuclear restricted, type II nuclear hormone receptors can be enhanced by a family of intracellular transport proteins, the fatty acid binding proteins (FABPs). We find that the transcriptional activity of the GR can be altered by a sub-set of FABP family members dependent on the GR-ligand. The ability of some FABPs to selectively promote or limit the transcriptional activity of the GR in a ligand-dependent manner could facilitate the discovery of drugs that narrow GR activity to only the desired subset of therapeutically relevant genes.

Neurokinin 1 receptor signaling in endosomes mediates sustained nociception and is a viable therapeutic target for prolonged pain relief

Publisher: American Association for the Advancement of Science (AAAS)

Date: 31-05-2017

DOI: 10.1126/SCITRANSLMED.AAL3447

Abstract: Therapeutic targeting of the neurokinin 1 receptor in endosomes provides efficacious and prolonged pain relief.

HN, N, Cα and Cβ assignments of the two periplasmic domains of Neisseria meningitidis DsbD

Publisher: Springer Science and Business Media LLC

Date: 06-06-2017

DOI: 10.1007/S12104-017-9743-X

Abstract: DsbD is a disulfide bond reductase present in the inner membrane of many Gamma-Proteobacteria. In the human pathogen Neisseria meningitidis, DsbD is required for viability and represents a potential target for the development of antibiotics. Here we report the chemical shift assignments (H

Distinct binding properties of TIAR RRMs and linker region

Publisher: Informa UK Limited

Date: 04-2013

DOI: 10.4161/RNA.24341

Fluoromethylketone-fragment conjugates designed as covalent modifiers of EcDsbA are atypical substrates

Publisher: American Chemical Society (ACS)

Date: 17-02-2022

DOI: 10.26434/CHEMRXIV-2022-262LH

Abstract: Disulfide bond protein A (DsbA) is an oxidoreductase enzyme that catalyzes the formation of disulfide bonds in Gram-negative bacteria. In Escherichia coli, DsbA (EcDsbA) is essential for bacterial virulence, thus inhibitors have the potential to act as antivirulence agents. A fragment-based screen was conducted against EcDsbA and herein we describe the development of a series of compounds based on a phenylthiophene hit identified from the screen. A novel thiol reactive and “clickable” ethynylfluoromethylketone was designed for reaction with azide-functionalized fragments to enable rapid and versatile attachment to a range of fragments. The resulting fluoromethylketone conjugates showed selectivity for reaction with the active site thiol of EcDsbA, however unexpectedly, turnover of the covalent adduct was observed. A mechanism for this turnover was investigated and proposed which may have wider ramifications for covalent reactions with dithiol-disulfide oxidoreducatases.

Self-micellization of gemfibrozil 1-0- acyl glucuronide in aqueous solution

Publisher: Springer Science and Business Media LLC

Date: 2003

DOI: 10.1023/A:1022672608657

The first total synthesis and solution structure of a polypeptin, PE2, a cyclic lipopeptide with broad spectrum antibiotic activity

Publisher: Royal Society of Chemistry (RSC)

Date: 2017

DOI: 10.1039/C7OB01493G

Abstract: The synthesis and NMR structure of a polypeptin, a depsipeptide that shows anti-bacterial activity against drug resistant bacteria has been achieved.

Application of Fragment‐Based Screening to the Design of Inhibitors of Escherichia coli DsbA

Publisher: Wiley

Date: 30-12-2014

DOI: 10.1002/ANIE.201410341

Abstract: The thiol-disulfide oxidoreductase enzyme DsbA catalyzes the formation of disulfide bonds in the periplasm of Gram-negative bacteria. DsbA substrates include proteins involved in bacterial virulence. In the absence of DsbA, many of these proteins do not fold correctly, which renders the bacteria avirulent. Thus DsbA is a critical mediator of virulence and inhibitors may act as antivirulence agents. Biophysical screening has been employed to identify fragments that bind to DsbA from Escherichia coli. Elaboration of one of these fragments produced compounds that inhibit DsbA activity in vitro. In cell-based assays, the compounds inhibit bacterial motility, but have no effect on growth in liquid culture, which is consistent with selective inhibition of DsbA. Crystal structures of inhibitors bound to DsbA indicate that they bind adjacent to the active site. Together, the data suggest that DsbA may be amenable to the development of novel antibacterial compounds that act by inhibiting bacterial virulence.

Sent packing: protein engineering generates a new crystal form ofPseudomonas aeruginosaDsbA1 with increased catalytic surface accessibility

Publisher: International Union of Crystallography (IUCr)

Date: 26-11-2015

DOI: 10.1107/S1399004715018519

Abstract: Pseudomonas aeruginosa is an opportunistic human pathogen for which new antimicrobial drug options are urgently sought. P. aeruginosa disulfide-bond protein A1 (PaDsbA1) plays a pivotal role in catalyzing the oxidative folding of multiple virulence proteins and as such holds great promise as a drug target. As part of a fragment-based lead discovery approach to PaDsbA1 inhibitor development, the identification of a crystal form of PaDsbA1 that was more suitable for fragment-soaking experiments was sought. A previously identified crystallization condition for this protein was unsuitable, as in this crystal form of PaDsbA1 the active-site surface loops are engaged in the crystal packing, occluding access to the target site. A single residue involved in crystal-packing interactions was substituted with an amino acid commonly found at this position in closely related enzymes, and this variant was successfully used to generate a new crystal form of PaDsbA1 in which the active-site surface is more accessible for soaking experiments. The PaDsbA1 variant displays identical redox character and in vitro activity to wild-type PaDsbA1 and is structurally highly similar. Two crystal structures of the PaDsbA1 variant were determined in complex with small molecules bound to the protein active site. These small molecules (MES, glycerol and ethylene glycol) were derived from the crystallization or cryoprotectant solutions and provide a proof of principle that the reported crystal form will be amenable to co-crystallization and soaking with small molecules designed to target the protein active-site surface.

Structural basis for the inhibition of poxvirus assembly by the antibiotic rifampicin

Publisher: Proceedings of the National Academy of Sciences

Date: 08-2018

DOI: 10.1073/PNAS.1810398115

Abstract: Most antibiotics do not interfere with viral infections. Rif icin is a notable exception, as it inhibits several poxviruses, including the causative agent of smallpox. However, the inhibition of viral assembly is unrelated to the antibacterial activity of rif icin against microbial RNA polymerases. Here, we reveal how the antibiotic prevents the recruitment of an essential scaffolding protein to nascent viral membranes. Based on these results, we provide a structural model of membrane assembly that is distinct from budding through cellular membranes and is most likely conserved in many large DNA viruses. Together, the mechanism of membrane assembly and structural models provide avenues to develop broad spectrum inhibitors against human and animal poxviruses.

Comparative Sequence, Structure and Redox Analyses of Klebsiella pneumoniae DsbA Show That Anti-Virulence Target DsbA Enzymes Fall into Distinct Classes

Publisher: Public Library of Science (PLoS)

Date: 14-11-2013

DOI: 10.1371/JOURNAL.PONE.0080210

Fatty acid-binding protein 5 at the blood–brain barrier regulates endogenous brain docosahexaenoic acid levels and cognitive function

Publisher: Society for Neuroscience

Date: 16-11-2016

DOI: 10.1523/JNEUROSCI.1583-16.2016

Abstract: Fatty acid-binding protein 5 (FABP5) at the blood–brain barrier contributes to the brain uptake of docosahexaenoic acid (DHA), a blood-derived polyunsaturated fatty acid essential for maintenance of cognitive function. Given the importance of DHA in cognition, the aim of this study was to investigate whether deletion of FABP5 results in cognitive dysfunction and whether this is associated with reduced brain endothelial cell uptake of exogenous DHA and subsequent attenuation in the brain levels of endogenous DHA. Cognitive function was assessed in male and female FABP5 +/+ and FABP5 −/− mice using a battery of memory paradigms. FABP5 −/− mice exhibited impaired working memory and short-term memory, and these cognitive deficits were associated with a 14.7 ± 5.7% reduction in endogenous brain DHA levels. The role of FABP5 in the blood–brain barrier transport of DHA was assessed by measuring 14 C-DHA uptake into brain endothelial cells and capillaries isolated from FABP5 +/+ and FABP5 −/− mice. In line with a crucial role of FABP5 in the brain uptake of DHA, 14 C-DHA uptake into brain endothelial cells and brain capillaries of FABP5 −/− mice was reduced by 48.4 ± 14.5% and 14.0 ± 4.2%, respectively, relative to those of FABP5 +/+ mice. These results strongly support the hypothesis that FABP5 is essential for maintaining brain endothelial cell uptake of DHA, and that cognitive deficits observed in FABP5 −/− mice are associated with reduced CNS access of DHA. SIGNIFICANCE STATEMENT Genetic deletion of fatty acid-binding protein 5 (FABP5) in mice reduces uptake of exogenous docosahexaenoic acid (DHA) into brain endothelial cells and brain capillaries and reduces brain parenchymal levels of endogenous DHA. Therefore, FABP5 in the brain endothelial cell is a crucial contributor to the brain levels of DHA. Critically, lowered brain DHA levels in FABP5 −/− mice occurred in tandem with cognitive deficits in a battery of memory paradigms. This study provides evidence of a critical role for FABP5 in the maintenance of cognitive function via regulating the brain uptake of DHA, and suggests that upregulation of FABP5 in neurodegenerative diseases, where brain DHA levels are possibly diminished (e.g., Alzheimer's disease), may provide a novel therapeutic approach for restoring cognitive function.

Three-dimensional aerogel based on in-situ growth of 1T-MoS2 on functionalized hierarchical porous carbon/reduced graphene oxide for energy storage

Publisher: Elsevier BV

Date: 03-2020

DOI: 10.1016/J.APSUSC.2019.144811

Molecular dynamics of Poly(L-lysine) dendrimers with naphthalene disulfonate caps

Publisher: American Chemical Society (ACS)

Date: 10-03-2009

DOI: 10.1021/MA802154E

Rapid Elaboration of Fragments into Leads Applied to Bromodomain-3 Extra-Terminal Domain

Publisher: American Chemical Society (ACS)

Date: 18-04-2023

DOI: 10.1021/ACS.JMEDCHEM.3C00137

Conformational Selection of Inhibitors and Substrates by Proteolytic Enzymes:  Implications for Drug Design and Polypeptide Processing

Publisher: American Chemical Society (ACS)

Date: 11-03-2000

DOI: 10.1021/JM990315T

Abstract: Inhibitors of proteolytic enzymes (proteases) are emerging as prospective treatments for diseases such as AIDS and viral infections, cancers, inflammatory disorders, and Alzheimer's disease. Generic approaches to the design of protease inhibitors are limited by the unpredictability of interactions between, and structural changes to, inhibitor and protease during binding. A computer analysis of superimposed crystal structures for 266 small molecule inhibitors bound to 48 proteases (16 aspartic, 17 serine, 8 cysteine, and 7 metallo) provides the first conclusive proof that inhibitors, including substrate analogues, commonly bind in an extended beta-strand conformation at the active sites of all these proteases. Representative superimposed structures are shown for (a) multiple inhibitors bound to a protease of each class, (b) single inhibitors each bound to multiple proteases, and (c) conformationally constrained inhibitors bound to proteases. Thus inhibitor/substrate conformation, rather than sequence/composition alone, influences protease recognition, and this has profound implications for inhibitor design. This conclusion is supported by NMR, CD, and binding studies for HIV-1 protease inhibitors/substrates which, when preorganized in an extended conformation, have significantly higher protease affinity. Recognition is dependent upon conformational equilibria since helical and turn peptide conformations are not processed by proteases. Conformational selection explains the resistance of folded/structured regions of proteins to proteolytic degradation, the susceptibility of denatured proteins to processing, and the higher affinity of conformationally constrained 'extended' inhibitors/substrates for proteases. Other approaches to extended inhibitor conformations should similarly lead to high-affinity binding to a protease.

Characterization of the N-Methyltransferase Activities of the Multifunctional Polypeptide Cyclosporin Synthetase

Publisher: Elsevier BV

Date: 04-2011

DOI: 10.1016/J.CHEMBIOL.2011.01.017

Abstract: This study demonstrates a critical role for N-methylation in cyclosporin biosynthesis and maintenance of the biologically active cyclosporin conformation. The structural requirements for the AdoMet binding to CySyn were defined. N-methylation of specific amide positions in the cyclosporin backbone is critical for the complete assembly and cyclization of the cyclosporin peptide. A maximum of two desmethyl positions is tolerated before peptide assembly stalls. Subinhibitory concentrations of AdoMet analogs directed peptide assembly towards cyclosporins with less than seven N-methylated amide bonds. Molecular modeling and nuclear magnetic resonance analyses indicate that N-methylation of specific amide bond positions in the cyclosporin backbone is mandatory for the formation of a product-like conformation and recognition by the acceptor site of the downstream peptide bond forming C-domain.

Three-Dimensional Structure in Solution of the Polypeptide Cardiac Stimulant Anthopleurin-A

Publisher: American Chemical Society (ACS)

Date: 21-03-1995

DOI: 10.1021/BI00011A036

Abstract: The three-dimensional structure in aqueous solution of the 49-residue polypeptide anthopleurin-A (AP-A), from the sea anemone Anthopleura xanthogrammica, has been determined from 1H NMR data. A restraint set consisting of 411 interproton distance restraints inferred from NOEs and 19 backbone and 13 side chain dihedral angle restraints from spin-spin coupling constants, as well as 15 lower bound restraints based on the absence of NOEs in the spectra, was used as input for distance geometry calculations in DIANA and simulated annealing and restrained energy minimization in X-PLOR. Stereospecific assignments for 12 beta-methylene pairs were also included. The final set of 20 structures had mean pairwise rms differences over the whole molecule of 2.04 A for the backbone heavy atoms (N, C alpha, and C) and 2.59 A for all heavy atoms. For the well-defined region encompassing residues 2-7 and 17-49, the corresponding values were 0.82 and 1.27 A, respectively. AP-A adopts a compact structure consisting of four short strands of antiparallel beta-sheet (residues 2-4, 20-23, 34-37, and 45-48) connected by three loops. The first loop commences with a type I beta-turn which includes two important Asp residues this loop is the least well-defined region of the protein, although a beta-turn involving residues 13-16 is observed in nearly half the structures. The loop linking the second and third strands is constrained by the 29-47 disulfide bond and contains two well-defined beta-turns, while the third loop contains the Gly40-Pro41 sequence, which has been identified previously as the site of cis-trans isomerism. The carboxylate group of Asp7 is close to the epsilon-ammonium group of Lys37, suggesting that they may form a salt bridge. A pH titration monitored by 2D NMR supports this by showing that Asp7 has a low pKa. It is proposed that this region of the molecule and the nearby residues Asp9 and His39 form part of the molecular surface which interacts with the mammalian cardiac sodium channel.

Fatty acid-binding proteins 1 and 2 differentially modulate the activation of peroxisome proliferator-activated receptor alpha in a ligand-selective manner

Publisher: Elsevier BV

Date: 05-2015

DOI: 10.1074/JBC.M114.605998

Examination of the Role of Intestinal Fatty Acid-Binding Protein in Drug Absorption Using a Parallel Artificial Membrane Permeability Assay

Publisher: Elsevier BV

Date: 04-2007

DOI: 10.1016/J.CHEMBIOL.2007.03.009

Abstract: Transcellular diffusion across the absorptive epithelial cells (enterocytes) of the small intestine is the main route of absorption for most orally administered drugs. The process by which lipophilic compounds transverse the aqueous environment of the cytoplasm, however, remains poorly defined. In the present study, we have identified a structurally erse group of lipophilic drugs that display low micromolar binding affinities for a cytosolic lipid-binding protein - intestinal fatty acid-binding protein (I-FABP). Binding to I-FABP significantly enhanced the transport of lipophilic drug molecules across a model membrane, and the degree of transport enhancement was related to both drug lipophilicity and I-FABP binding affinity. These data suggest that intracellular lipid-binding proteins such as I-FABP may enhance the membrane transport of lipophilic xenobiotics and facilitate drug access to the enterocyte cytoplasm and cytoplasmic organelles.

Molecular dynamics of variegated polyamide dendrimers

Publisher: American Chemical Society (ACS)

Date: 18-03-2009

DOI: 10.1021/MA8021579

Crystal structure of the HIV-1 integrase core domain in complex with sucrose reveals details of an allosteric inhibitory binding site

Publisher: Wiley

Date: 12-03-2010

DOI: 10.1016/J.FEBSLET.2010.03.016

Abstract: HIV integrase (IN) is an essential enzyme in HIV replication and an important target for drug design. IN has been shown to interact with a number of cellular and viral proteins during the integration process. Disruption of these important interactions could provide a mechanism for allosteric inhibition of IN. We present the highest resolution crystal structure of the IN core domain to date. We also present a crystal structure of the IN core domain in complex with sucrose which is bound at the dimer interface in a region that has previously been reported to bind integrase inhibitors.

Structure of a putative ancestral protein encoded by a single sequence repeat from a multidomain proteinase inhibitor gene fromNicotiana alata

Publisher: Elsevier BV

Date: 07-1999

DOI: 10.1016/S0969-2126(99)80103-8

Abstract: The ornamental tobacco Nicotiana alata produces a series of proteinase inhibitors (PIs) that are derived from a 43 kDa precursor protein, NaProPI. NaProPI contains six highly homologous repeats that fold to generate six separate structural domains, each corresponding to one of the native PIs. An unusual feature of NaProPI is that the structural domains lie across adjacent repeats and that the sixth PI domain is generated from fragments of the first and sixth repeats. Although the homology of the repeats suggests that they may have arisen from gene duplication, the observed folding does not appear to support this. This study of the solution structure of a single NaProPI repeat (aPI1) forms a basis for unravelling the mechanism by which this protein may have evolved. The three-dimensional structure of aPI1 closely resembles the triple-stranded antiparallel beta sheet observed in each of the native PIs. The five-residue sequence Glu-Glu-Lys-Lys-Asn, which forms the linker between the six structural domains in NaProPI, exists as a disordered loop in aPI1. The presence of this loop in aPI1 results in a loss of the characteristically flat and disc-like topography of the native inhibitors. A single repeat from NaProPI is capable of folding into a compact globular domain that displays native-like PI activity. Consequently, it is possible that a similar single-domain inhibitor represents the ancestral protein from which NaProPI evolved.

Δ‐Myrtoxin‐Mp1a is a Helical Heterodimer from the Venom of the Jack Jumper Ant that has Antimicrobial, Membrane‐Disrupting, and Nociceptive Activities

Publisher: Wiley

Date: 13-06-2017

DOI: 10.1002/ANIE.201703360

Abstract: Δ-Myrtoxin-Mp1a (Mp1a), a 49-residue heterodimeric peptide from the venom of Myrmecia pilosula, comprises a 26-mer A chain and a 23-mer B chain connected by two disulfide bonds in an antiparallel arrangement. Combination of the in idual synthetic chains through aerial oxidation remarkably resulted in the self-assembly of Mp1a as a homogenous product without the need for directed disulfide-bond formation. NMR analysis revealed a well-defined, unique structure containing an antiparallel α-helix pair. Dual polarization interferometry (DPI) analysis showed strong interaction with supported lipid bilayers and insertion within the bilayers. Mp1a caused non-specific Ca

Isolation, Solution Structure, and Insecticidal Activity of Kalata B2, a Circular Protein with a Twist:  Do Möbius Strips Exist in Nature?^,

Publisher: American Chemical Society (ACS)

Date: 24-12-2004

DOI: 10.1021/BI047837H

Abstract: A large number of macrocyclic miniproteins with erse biological activities have been isolated from the Rubiaceae, Violaceae, and Cucurbitaceae plant families in recent years. Here we report the three-dimensional structure determined using (1)H NMR spectroscopy and demonstrate potent insecticidal activity for one of these peptides, kalata B2. This peptide is one of the major components of an extract from the leaves of the plant Oldenlandia affinis. The structure consists of a distorted triple-stranded beta-sheet and a cystine knot arrangement of the disulfide bonds and is similar to those described for other members of the cyclotide family. The unique cyclic and knotted nature of these molecules makes them a fascinating ex le of topologically complex proteins. Examination of the sequences reveals that they can be separated into two subfamilies, one of which contains a larger number of positively charged residues and has a bracelet-like circularization of the backbone. The second subfamily contains a backbone twist due to a cis-peptidyl-proline bond and may conceptually be regarded as a molecular Mobius strip. Kalata B2 is the second putative member of the Mobius cyclotide family to be structurally characterized and has a cis-peptidyl-proline bond, thus validating the suggested name for this subfamily of cyclotides. The observation that kalata B2 inhibits the growth and development of Helicoverpa armigera larvae suggests a role for the cyclotides in plant defense. A comparison of the sequences and structures of kalata B1 and B2 provides insight into the biological activity of these peptides.

Promiscuous 2-Aminothiazoles (PrATs): A Frequent Hitting Scaffold

Publisher: American Chemical Society (ACS)

Date: 16-01-2015

DOI: 10.1021/JM501402X

Abstract: We have identified a class of molecules, known as 2-aminothiazoles (2-ATs), as frequent-hitting fragments in biophysical binding assays. This was exemplified by 4-phenylthiazol-2-amine being identified as a hit in 14/14 screens against a erse range of protein targets, suggesting that this scaffold is a poor starting point for fragment-based drug discovery. This prompted us to analyze this scaffold in the context of an academic fragment library used for fragment-based drug discovery (FBDD) and two larger compound libraries used for high-throughput screening (HTS). This analysis revealed that such "promiscuous 2-aminothiazoles" (PrATs) behaved as frequent hitters under both FBDD and HTS settings, although the problem was more pronounced in the fragment-based studies. As 2-ATs are present in known drugs, they cannot necessarily be deemed undesirable, but the combination of their promiscuity and difficulties associated with optimizing them into a lead compound makes them, in our opinion, poor scaffolds for fragment libraries.

Synthesis and photophysical studies of fluorenone-armed porphyrin arrays

Publisher: Elsevier BV

Date: 09-2016

DOI: 10.1016/J.TET.2016.07.028

Synthesis of unsymmetrical 1,1′-disubstituted bis(1,2,3-triazole)s using monosilylbutadiynes

Publisher: American Chemical Society (ACS)

Date: 12-01-2011

DOI: 10.1021/OL102852Z

Abstract: Bis(1,2,3-triazole)s have attracted recent interest as coordinating ligands for transition metals. Here we report a rapid, modular method for the synthesis of 1,1'-disubstituted-4,4'-linked unsymmetrical bis(1,2,3-triazole)s. The method employs sequential copper catalyzed azide-alkyne cycloaddition and deprotection steps on a monosilylbutadiyne. TMS (trimethylsilyl) and TIPS (triisopropylsilyl) were both investigated with TIPS being the preferred protecting group due to increased stability. The reactions were amenable to one-pot synthesis, and an optimized one-pot, three-step procedure was developed.

Structural and biochemical insights into the disulfide reductase mechanism of DsbD, an essential enzyme for neisserial pathogens

Publisher: Elsevier BV

Date: 10-2018

DOI: 10.1074/JBC.RA118.004847

Targeting virulence not viability in the search for future antibacterials

Publisher: Wiley

Date: 20-01-2015

DOI: 10.1111/BCP.12356

Blind man's bluff - Elaboration of fragment hits in the absence of structure for the development of antitrypsin deficiency inhibitors

Publisher: CSIRO Publishing

Date: 2013

DOI: 10.1071/CH13290

Abstract: The three pillars of rational drug design from a fragment library are an efficient screen, a robust assay, and atomic-resolution structures of the protein–ligand complexes. However, not all targets are amenable to structure determination by X-ray crystallography or NMR spectroscopy. In particular, targets involved in diseases of protein misfolding are inherently intractable. In the absence of structures, we are blind. However, the lack of structural information need not preclude the use of fragment-based approaches. The use of appropriate NMR techniques can enable us to detect the effects of protein binding on ligand resonances. In our efforts to identify compounds that affect the kinetics of α1-antitrypsin misfolding, we have used saturation transfer difference NMR spectroscopy to detect hits from mixtures of compounds in a fragment library. In the absence of structures, the initial challenge is three-fold: to (1) distinguish between binding sites (2) evaluate the relative affinities of hits and (3) advance them to the stage where activity can be detected in biological assays. We largely achieved these aims by the use of Carr–Purcell–Meiboom–Gill NMR competition experiments that detect differential relaxation of the ligand on protein binding.

Structure–Activity Studies of β-Hairpin Peptide Inhibitors of the Plasmodium falciparum AMA1–RON2 Interaction

Publisher: Elsevier BV

Date: 10-2016

DOI: 10.1016/J.JMB.2016.07.001

Abstract: The interaction between apical membrane antigen 1 (AMA1) and rhoptry neck protein 2 (RON2) plays a key role in the invasion of red blood cells by Plasmodium parasites. Disruption of this critical protein-protein interaction represents a promising avenue for antimalarial drug discovery. In this work, we exploited a 13-residue β-hairpin based on the C-terminal loop of RON2 to probe a conserved binding site on Plasmodium falciparum AMA1. A series of mutations was synthetically engineered into β-hairpin peptides to establish structure-activity relationships. The best mutations improved the binding affinity of the β-hairpin peptide by ~7-fold for 3D7 AMA1 and ~14-fold for FVO AMA1. We determined the crystal structures of several β-hairpin peptides in complex with AMA1 in order to define the structural features and specific interactions that contribute to improved binding affinity. The same mutations in the longer RON2sp2 peptide (residues 2027-2055 of RON2) increased the binding affinity by >30-fold for 3D7 and FVO AMA1, producing K

The interaction of lipophilic drugs with intestinal fatty acid-binding protein

Publisher: Elsevier BV

Date: 05-2005

DOI: 10.1074/JBC.M410193200

Fatty Acid Binding Proteins Expressed at the Human Blood–Brain Barrier Bind Drugs in an Isoform-Specific Manner

Publisher: Springer Science and Business Media LLC

Date: 07-08-2015

DOI: 10.1007/S11095-015-1764-5

Abstract: To examine the expression of fatty acid binding proteins (FABPs) at the human blood-brain barrier (BBB) and to assess their ability to bind lipophilic drugs. mRNA and protein expression of FABP subtypes in immortalized human brain endothelial (hCMEC/D3) cells were examined by RT-qPCR and Western blot, respectively. FABPs that were found in hCMEC/D3 cells (hFABPs) were recombinantly expressed and purified from Escherichia coli C41(DE3) cells. Drug binding to these hFABPs was assessed using a fluorescence assay, which measured the ability of a panel of lipophilic drugs to displace the fluorescent probe compound 1-anilinonaphthalene-8-sulfonic acid (ANS). hFABP3, 4 and 5 were expressed in hCMEC/D3 cells at the mRNA and protein level. The competitive ANS displacement assay demonstrated that, in general, glitazones preferentially bound to hFABP5 (Ki: 1.0-28 μM) and fibrates and fenamates preferentially bound to hFABP4 (Ki: 0.100-17 μM). In general, lipophilic drugs appeared to show weaker affinities for hFABP3 relative to hFABP4 and hFABP5. No clear correlation was observed between the molecular structure or physicochemical properties of the drugs and their ability to displace ANS from hFABP3, 4 and 5. hFABP3, 4 and 5 are expressed at the human BBB and bind differentially to a erse range of lipophilic drugs. The unique expression and binding patterns of hFABPs at the BBB may therefore influence drug disposition into the brain.

Laboratory testing for activated protein C resistance: rivaroxaban induced interference and a comparative evaluation of andexanet alfa and DOAC Stop to neutralise interference

Publisher: Walter de Gruyter GmbH

Date: 03-03-2020

DOI: 10.1515/CCLM-2019-1160

Abstract: Investigation of hemostasis is problematic when patients are on anticoagulant therapy. Rivaroxaban especially causes substantial interference, extending many clot-based tests, thereby leading to false positive or negative events. In particular, rivaroxaban affects some assays for activated protein C resistance (APCR). We assessed, in an international setting, cross laboratory (n = 31) testing using four s les to evaluate rivaroxaban induced interference in APCR testing, and whether this interference could be neutralised. The s les comprised: (A) pool of normal plasma (APCR-negative control) (B) this normal pool spiked with rivaroxaban (200 ng/mL) to create rivaroxaban-induced interference (potential ‘false’ positive APCR event s le) (C) the rivaroxaban s le subsequently treated with a commercial direct oral anticoagulant ‘DOAC-neutraliser’ (DOAC Stop), or (D) treated with andexanet alfa (200 μg/mL). Testing was performed blind to s le type. The rivaroxaban-spiked s le generated false positive APCR results for some, but unexpectedly not most APCR-tests. The s le treated with DOAC Stop evidenced a correction in the rivaroxaban-affected APCR assays, and did not otherwise adversely affect the rivaroxaban ‘unaffected’ APCR assays. The andexanet alfa-treated s le did not evidence correction of the false positive APCR, and instead unexpectedly exacerbated false positive APCR status with many tests. DOAC Stop was able to neutralise any APCR interference induced by rivaroxaban. In contrast, andexanet alfa did not negate such interference, and instead unexpectedly created more false-positive APCR events.

Fragment-Based Design of Ligands Targeting a Novel Site on the Integrase Enzyme of Human Immunodeficiency Virus1

Publisher: Wiley

Date: 16-12-2011

DOI: 10.1002/CMDC.201000483

Interrogating Fragments Using a Protein Thermal Shift Assay

Publisher: CSIRO Publishing

Date: 2013

DOI: 10.1071/CH13279

Abstract: Protein thermal shift is a relatively rapid and inexpensive technique for the identification of low molecular weight compound interactions with protein targets. An increase in the melting temperature of the target protein in the presence of a test ligand is indicative of a promising ligand–protein interaction. Due to its simplicity, protein thermal shift is an attractive method for screening libraries and validating hits in drug discovery programs. The methodology has been used successfully in high throughput screens of small molecule libraries, and its application has been extended to report on protein–drug-like-fragment interactions. Here, we review how protein thermal shift has been employed recently in fragment-based drug discovery (FBDD) efforts, and highlight its application to protein–protein interaction targets. Multiple validation of fragment hits by independent means is paramount to ensure efficient and economical progress in a FBDD c aign. We discuss the applicability of thermal shift assays in this light, and discuss more generally what one does when orthogonal approaches disagree.

Controlled Construction of Cyclic d / l Peptide Nanorods

Publisher: Wiley

Date: 05-12-2018

DOI: 10.1002/ANGE.201811910

Small Molecule Inhibitors of Disulfide Bond Formation by the Bacterial DsbA–DsbB Dual Enzyme System

Publisher: American Chemical Society (ACS)

Date: 27-01-2015

DOI: 10.1021/CB500988R

Abstract: The DsbA:DsbB redox machinery catalyzes disulfide bond formation in secreted proteins and is required for bacterial virulence factor assembly. Both enzymes have been identified as targets for antivirulence drugs. Here, we report synthetic analogues of ubiquinone (dimedone derivatives) that inhibit disulfide bond formation (IC50∼1 μM) catalyzed by E. coli DsbA:DsbB. The mechanism involves covalent modification of a single free cysteine leaving other cysteines unmodified. A vinylogous anhydride in each inhibitor is cleaved by the thiol, which becomes covalently modified to a thioester by a propionyl substituent. Cysteines and lysines on DsbA and DsbB and a nonredox enzyme were modified in a manner that implies some specificity. Moreover, human thioredoxin was not inhibited under the same conditions that inhibited EcDsbA. This proof of concept work uses small molecules that target specific cysteines to validate the DsbA and DsbB dual enzyme system as a viable and potentially druggable antivirulence target.

NMR fragment screening reveals a novel small molecule binding site near the catalytic surface of the disulfide–dithiol oxidoreductase enzyme DsbA from Burkholderia pseudomallei

Publisher: Springer Science and Business Media LLC

Date: 06-08-2020

DOI: 10.1007/S10858-020-00339-5

Small heat-shock proteins interact with a flanking domain to suppress polyglutamine aggregation

Publisher: Proceedings of the National Academy of Sciences

Date: 19-05-2010

DOI: 10.1073/PNAS.0914773107

Abstract: Small heat-shock proteins (sHsps) are molecular chaperones that play an important protective role against cellular protein misfolding by interacting with partially unfolded proteins on their off-folding pathway, preventing their aggregation. Polyglutamine (polyQ) repeat expansion leads to the formation of fibrillar protein aggregates and neuronal cell death in nine diseases, including Huntington disease and the spinocerebellar ataxias (SCAs). There is evidence that sHsps have a role in suppression of polyQ-induced neurodegeneration for ex le, the sHsp alphaB-crystallin (αB-c) has been identified as a suppressor of SCA3 toxicity in a Drosophila model. However, the molecular mechanism for this suppression is unknown. In this study we tested the ability of αB-c to suppress the aggregation of a polyQ protein. We found that αB-c does not inhibit the formation of SDS-insoluble polyQ fibrils. We further tested the effect of αB-c on the aggregation of ataxin-3, a polyQ protein that aggregates via a two-stage aggregation mechanism. The first stage involves association of the N-terminal Josephin domain followed by polyQ-mediated interactions and the formation of SDS-resistant mature fibrils. Our data show that αB-c potently inhibits the first stage of ataxin-3 aggregation however, the second polyQ-dependent stage can still proceed. By using NMR spectroscopy, we have determined that αB-c interacts with an extensive region on the surface of the Josephin domain. These data provide an ex le of a domain/region flanking an amyloidogenic sequence that has a critical role in modulating aggregation of a polypeptide and plays a role in the interaction with molecular chaperones to prevent this aggregation.

Determinants of Proteolysis and Cell-Binding for the Shigella flexneri Cytotoxin, SigA

Publisher: Springer Science and Business Media LLC

Date: 22-08-2015

DOI: 10.1007/S00284-015-0893-8

Abstract: Shigella flexneri secretes an enterotoxic, SPATE family autotransporter (AT), SigA, which has cytopathic activity towards cultured epithelial cells. Its cytopathic activity is due to its ability to degrade the cytoskeletal protein, α-fodrin. The mechanisms by which AT toxins target cells and tissues differ and the details of how SigA acts are not known. In the current study, the determinants of proteolysis and cell-targeting for SigA were determined. We demonstrate that the SigA passenger or α-domain consists of two functionally distinct domains, designated α1 and α2, which are sufficient to specify proteolytic and cell-binding activities, respectively.

The Structural Impact of a Polyglutamine Tract Is Location-Dependent

Publisher: Elsevier BV

Date: 12-2008

DOI: 10.1529/BIOPHYSJ.108.138487

Applications of NMR Spectroscopy in FBDD

Publisher: Springer International Publishing

Date: 2018

DOI: 10.1007/978-3-319-28388-3_127

Inhibitors of BCATm: A Tough Nut To Crack

Publisher: American Chemical Society (ACS)

Date: 04-09-2015

DOI: 10.1021/ACS.JMEDCHEM.5B01244

Abstract: Inhibitors of the mitochondrial branched chain aminotransferase may have therapeutic potential in the treatment of diet-induced obesity and dyslipidemia. To explore the pharmacology of this metabolic pathway requires a potent and selective molecule that is well tolerated and has appropriate pharmacokinetic properties. The combination of fragment-based and high-throughput screening with structure-guided compound elaboration has yielded a tool compound that may serve as a chemical probe of this pathway.

The solution structure of C1-T1, a two-domain proteinase inhibitor derived from a circular precursor protein from Nicotiana alata11Edited by P. E. Wright

Publisher: Elsevier BV

Date: 02-2001

DOI: 10.1006/JMBI.2000.4318

An improved method for the purification of rat liver-type fatty acid binding protein from Escherichia coli

Publisher: Elsevier BV

Date: 11-2005

DOI: 10.1016/J.PEP.2005.04.006

Abstract: Rat liver fatty acid binding protein (L-FABP) was efficiently expressed in Escherichia coli and purified to homogeneity. The cDNA encoding L-FABP was ligated into the pTrc99A expression vector and expressed by induction with isopropyl-beta-d-thiogalactopyranoside under the control of the P(trc) promoter. Following an 18 h induction period, L-FABP constituted approximately 3% of the cytosolic protein. The protein could be purified to electrophoretic homogeneity (silver-stained polyacrylamide gel detection) by ammonium sulfate fractionation (65% saturation) of the soluble bacterial lysate followed by the chromatographic sequence of anion-exchange-->hydrophobic interaction-->anion-exchange chromatography. The recombinant protein displayed an isoelectric point of 7.0 and cross-reactivity with rabbit anti-(human L-FABP) polyclonal antibody. The ligand binding properties of the delipidated L-FABP were examined by titration with the fluorescent probe 1-anilino-8-naphthalene sulfonic acid and isothermal titration calorimetric analysis of oleic acid binding. The purified rat L-FABP displayed a binding stoichiometry of 2:1 (ANS:L-FABP) with dissociation constants (K(d)) of 1.7 and 15.5 microM for the high and low affinity binding sites, respectively. The K(d) values determined by ITC for oleic acid binding were 0.155 and 4.04 microM with a binding stoichiometry of approximately 2 mol of fatty acid/mol of protein. These physicochemical and binding properties are in agreement with those of L-FABP isolated from rat liver tissue.

Corrigendum: Fragment-Based Design of Ligands Targeting a Novel Site on the Integrase Enzyme of Human Immunodeficiency Virus1

Publisher: Wiley

Date: 25-03-2011

DOI: 10.1002/CMDC.201190013

Conserved features in TamA enable interaction with TamB to drive the activity of the translocation and assembly module

Publisher: Springer Science and Business Media LLC

Date: 05-08-2015

DOI: 10.1038/SREP12905

Abstract: The biogenesis of membranes from constituent proteins and lipids is a fundamental aspect of cell biology. In the case of proteins assembled into bacterial outer membranes, an overarching question concerns how the energy required for protein insertion and folding is accessed at this remote location of the cell. The translocation and assembly module (TAM) is a nanomachine that functions in outer membrane biogenesis and virulence in erse bacterial pathogens. Here we demonstrate the interactions through which TamA and TamB subunits dock to bridge the periplasm and unite the outer membrane aspects to the inner membrane of the bacterial cell. We show that specific functional features in TamA have been conserved through evolution, including residues surrounding the lateral gate and an extensive surface of the POTRA domains. Analysis by nuclear magnetic resonance spectroscopy and small angle X-ray scattering document the characteristic structural features of these POTRA domains and demonstrate rigidity in solution. Quartz crystal microbalance measurements pinpoint which POTRA domain specifically docks the TamB subunit of the nanomachine. We speculate that the POTRA domain of TamA functions as a lever arm in order to drive the activity of the TAM, assembling proteins into bacterial outer membranes.

Structure and Function of the Oxidoreductase DsbA1 from Neisseria meningitidis

Publisher: Elsevier BV

Date: 12-2009

DOI: 10.1016/J.JMB.2009.09.065

Abstract: Neisseria meningitidis encodes three DsbA oxidoreductases (NmDsbA1-NmDsbA3) that are vital for the oxidative folding of many membrane and secreted proteins, and these three enzymes are considered to exhibit different substrate specificities. This has led to the suggestion that each N. meningitidis DsbA (NmDsbA) may play a specialized role in different stages of pathogenesis however, the molecular and structural bases of the different roles of NmDsbAs are unclear. With the aim of determining the molecular basis for substrate specificity and how this correlates to pathogenesis, we undertook a biochemical and structural characterization of the three NmDsbAs. We report the 2.0-A-resolution crystal structure of the oxidized form of NmDsbA1, which adopted a canonical DsbA fold similar to that observed in the structures of NmDsbA3 and Escherichia coli DsbA (EcDsbA). Structural comparisons revealed variations around the active site and candidate peptide-binding region. Additionally, we demonstrate that all three NmDsbAs are strong oxidases with similar redox potentials however, they differ from EcDsbA in their ability to be reoxidized by E. coli DsbB. Collectively, our studies suggest that the small structural differences between the NmDsbA enzymes and EcDsbA are functionally significant and are the likely determinants of substrate specificity.

Characterization of two distinct modes of drug binding to human intestinal fatty acid binding protein

Publisher: American Chemical Society (ACS)

Date: 02-09-2014

DOI: 10.1021/CB5005178

Abstract: The aqueous cytoplasm of cells poses a potentially significant barrier for many lipophilic drugs to reach their sites of action. Fatty acid binding proteins (FABPs) bind to poorly water-soluble fatty acids (FAs) and lipophilic compounds and facilitate their intracellular transport. Several structures of FA in complex with FABPs have been described, but data describing the binding sites of other lipophilic ligands including drugs are limited. Here the environmentally sensitive fluorophores, 1-anilinonapthalene 8-sulfonic acid (ANS), and 11-dansylamino undecanoic acid (DAUDA) were used to investigate drug binding to human intestinal FABP (hIFABP). Most drugs that bound hIFABP were able to displace both ANS and DAUDA. A notable exception was ketorolac, a non-steroidal anti-inflammatory drug that bound to hIFABP and displaced DAUDA but failed to displace ANS. Isothermal titration calorimetry revealed that for the majority of ligands including FA, ANS, and DAUDA, binding to hIFABP was exothermic. In contrast, ketorolac binding to hIFABP was endothermic and entropy-driven. The X-ray crystal structure of DAUDA-hIFABP revealed a FA-like binding mode where the carboxylate of DAUDA formed a network of hydrogen bonds with residues at the bottom of the binding cavity and the dansyl group interacted with residues in the portal region. In contrast, NMR chemical shift perturbation (CSP) data suggested that ANS bound only toward the bottom of the hIFABP cavity, whereas ketorolac occupied only the portal region. The CSP data further suggested that ANS and ketorolac were able to bind simultaneously to hIFABP, consistent with the lack of displacement of ANS observed by fluorescence and supported by a model of the ternary complex. The NMR solution structure of the ketorolac-hIFABP complex therefore describes a newly characterized, hydrophobic ligand binding site in the portal region of hIFABP.

The binding interaction of synthetic ozonide antimalarials with natural and modified beta-cyclodextrins

Publisher: Elsevier BV

Date: 2006

DOI: 10.1002/JPS.20525

¹⁹F NMR as a Probe of Ligand Interactions with the iNOS Binding site of SPRY Domain‐Containing SOCS Box Protein 2

Publisher: Wiley

Date: 05-06-2014

DOI: 10.1111/CBDD.12355

Abstract: SPRY domain-containing SOCS box protein 2 (SPSB2) regulates inducible nitric oxide synthase (iNOS) by targeting it for proteasomal degradation. Inhibiting this interaction prolongs the intracellular lifetime of iNOS, leading in turn to enhanced killing of infectious pathogens such as bacteria and parasites. SPSB2 recognizes a linear motif (DINNN) in the disordered N-terminus of iNOS, and ligands that target the DINNN binding site on SPSB2 are potentially novel anti-infective agents. We have explored (19)F NMR as a means of probing ligand binding to SPSB2. All six Trp residues in SPSB2 were replaced with 5-fluorotryptophan (5-F-Trp) by utilizing a Trp auxotroph strain of Escherichia coli. The labeled protein was well folded and bound a DINNN-containing peptide with similar affinity to native SPSB2. Six well-resolved 5-F-Trp resonances were observed in the (19)F NMR spectrum and were assigned using site-directed mutagenesis. The (19)F resonance of W207 was significantly perturbed upon binding to DINNN-containing peptides. Other resonances were perturbed to a lesser extent although in a way that was sensitive to the composition of the peptide. Analogues of compounds identified in a fragment screen also perturbed the W207 resonance, confirming their binding to the iNOS peptide-binding site on SPSB2. (19)F NMR promises to be a valuable approach in developing inhibitors that bind to the DINNN binding site.

Synthesis and elaboration of N-methylpyrrolidone as an acetamide fragment substitute in bromodomain inhibition

Publisher: Elsevier BV

Date: 12-2019

DOI: 10.1016/J.BMC.2019.115157

Abstract: N-Methylpyrrolidone is a solvent molecule which has been shown to compete with acetyl-lysine-containing peptides for binding to bromodomains. From crystallographic studies, it has also been shown to closely mimic the acetamide binding motif in several bromodomains, but has not yet been directly pursued as a fragment in bromodomain inhibition. In this paper, we report the elaboration of N-methylpyrrolidone as a potential lead in fragment-based drug design. Firstly, N-methylpyrrolidone was functionalised to provide points for chemical elaboration. Then, the moiety was incorporated into analogues of the reported bromodomain inhibitor, Olinone. X-ray crystallography revealed that the modified analogues showed comparable binding affinity and structural mimicry to Olinone in the bromodomain binding site.

Design and Evaluation of the Performance of an NMR Screening Fragment Library

Publisher: CSIRO Publishing

Date: 2013

DOI: 10.1071/CH13280

Abstract: The design of a suitable library is an essential prerequisite to establish a fragment-based screening capability. Several pharmaceutical companies have described their approaches to establishing fragment libraries however there are few detailed reports of both design and analysis of performance for a fragment library maintained in an academic setting. Here we report our efforts towards the design of a fragment library for nuclear magnetic resonance spectroscopy-based screening, demonstrate the performance of the library through analysis of 14 screens, and present a comparison to previously reported fragment libraries.

Targeting Bacterial Dsb Proteins for the Development of Anti-Virulence Agents

Publisher: MDPI AG

Date: 16-07-2016

DOI: 10.3390/MOLECULES21070811

Solution Structure of BSTI:  A New Trypsin Inhibitor from Skin Secretions of Bombina bombina^,

Publisher: American Chemical Society (ACS)

Date: 17-03-2001

DOI: 10.1021/BI002623V

Abstract: The three-dimensional solution structure of BSTI, a trypsin inhibitor from the European frog Bombina bombina, has been solved using (1)H NMR spectroscopy. The 60 amino acid protein contains five disulfide bonds, which were unambiguously determined to be Cys (4--38), Cys (13--34), Cys (17--30), Cys (21--60), and Cys (40--54) by experimental restraints and subsequent structure calculations. The main elements of secondary structure are four beta-strands, arranged as two small antiparallel beta-sheets. The overall fold of BSTI is disk shaped and is characterized by the lack of a hydrophobic core. The presumed active site is located on a loop comprising residues 21--34, which is a relatively disordered region similar to that seen in many other protease inhibitors. However, the overall fold is different to other known protease inhibitors with the exception of a small family of inhibitors isolated from nematodes of the family Ascaris and recently also from the haemolymph of Apis mellifera. BSTI may thus be classified as a new member of this recently discovered family of protease inhibitors.

Fatty Acid-Binding Protein 5 Facilitates the Blood–Brain Barrier Transport of Docosahexaenoic Acid

Publisher: American Chemical Society (ACS)

Date: 30-10-2015

DOI: 10.1021/ACS.MOLPHARMACEUT.5B00580

Abstract: The brain has a limited ability to synthesize the essential polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA) from its omega-3 fatty acid precursors. Therefore, to maintain brain concentrations of this PUFA at physiological levels, plasma-derived DHA must be transported across the blood-brain barrier (BBB). While DHA is able to partition into the luminal membrane of brain endothelial cells, its low aqueous solubility likely limits its cytosolic transfer to the abluminal membrane, necessitating the requirement of an intracellular carrier protein to facilitate trafficking of this PUFA across the BBB. As the intracellular carrier protein fatty acid-binding protein 5 (FABP5) is expressed at the human BBB, the current study assessed the putative role of FABP5 in the brain endothelial cell uptake and BBB transport of DHA in vitro and in vivo, respectively. hFAPB5 was recombinantly expressed and purified from Escherichia coli C41(DE3) cells and the binding affinity of DHA to hFABP5 assessed using isothermal titration calorimetry. The impact of FABP5 siRNA on uptake of (14)C-DHA into immortalized human brain microvascular endothelial (hCMEC/D3) cells was assessed. An in situ transcardiac perfusion method was optimized in C57BL/6 mice and subsequently used to compare the BBB influx rate (Kin) of (14)C-DHA between FABP5-deficient (FABP5(-/-)) and wild-type (FABP5(+/+)) C57BL/6 mice. DHA bound to hFABP5 with an equilibrium dissociation constant of 155 ± 8 nM (mean ± SEM). FABP5 siRNA transfection decreased hCMEC/D3 mRNA and protein expression of FABP5 by 53.2 ± 5.5% and 44.8 ± 13.7%, respectively, which was associated with a 14.1 ± 2.7% reduction in (14)C-DHA cellular uptake. By using optimized conditions for the in situ transcardiac perfusion (a 1 min preperfusion (10 mL/min) followed by perfusion of (14)C-DHA (1 min)), the Kin of (14)C-DHA was 0.04 ± 0.01 mL/g/s. Relative to FABP5(+/+) mice, the Kin of (14)C-DHA decreased 36.7 ± 12.4% in FABP5(-/-) mice. This study demonstrates that FABP5 binds to DHA and is involved in the brain endothelial cell uptake and subsequent BBB transport of DHA, confirming the importance of this cytoplasmic carrier protein in the CNS exposure of this PUFA essential for neuronal function.

A novel two-chain proteinase inhibitor generated by circularization of a multidomain precursor protein

Publisher: Springer Science and Business Media LLC

Date: 06-1999

DOI: 10.1038/9293

Abstract: Female reproductive tissues of the ornamental tobacco amass high levels of serine proteinase inhibitors (PIs) for protection against pests and pathogens. These PIs are produced from a precursor protein composed of six repeats each with a protease reactive site. Here we show that proteolytic processing of the precursor generates five single-chain PIs and a remarkable two-chain inhibitor formed by disulfide-bond linkage of N- and C-terminal peptide fragments. Surprisingly, PI precursors adopt this circular structure regardless of the number of inhibitor domains, suggesting this bracelet-like conformation is characteristic of the widespread potato inhibitor II (Pot II) protein family.

Protein unfolding is essential for cleavage within the α-helix of a model protein substrate by the serine protease, thrombin

Publisher: Elsevier BV

Date: 03-2016

DOI: 10.1016/J.BIOCHI.2015.09.021

Abstract: Proteolysis has a critical role in transmitting information within a biological system and therefore an important element of biology is to determine the subset of proteins amenable to proteolysis. Until recently, it has been thought that proteases cleave native protein substrates only within solvent exposed loops, but recent evidence indicates that cleavage sites located within α-helices can also be cleaved by proteases, despite the conformation of this secondary structure being generally incompatible with binding into an active site of a protease. In this study, we address the mechanism by which a serine endopeptidase, thrombin, recognizes and cleaves a target sequence located within an α-helix. Thrombin was able to cleave a model substrate, protein G, within its α-helix when a suitable cleavage sequence for the enzyme was introduced into this region. However, structural data for the complex revealed that thrombin was not perturbing the structure of the α-helix, thus it was not destabilizing the helix in order to allow it to fit within its active site. This indicated that thrombin was only cleaving within the α-helix when it was in an unfolded state. In support of this, the introduction of destabilizing mutations within the protein increased the efficiency of cleavage by the enzyme. Our data suggest that a folded α-helix cannot be proteolytically cleaved by thrombin, but the species targeted are the unfolded conformations of the native state ensemble.

Fragment-based discovery of inhibitors of the bacterial DnaG-SSB interaction

Publisher: MDPI AG

Date: 22-02-2018

DOI: 10.3390/ANTIBIOTICS7010014

Fragment library screening identifies hits that bind to the non-catalytic surface of Pseudomonas aeruginosa DsbA1

Publisher: Public Library of Science (PLoS)

Date: 27-03-2017

DOI: 10.1371/JOURNAL.PONE.0173436

A simplified liquid chromatography assay for the quantitation of halofantrine and desbutylhalofantrine in plasma and identification of a degradation product of desbutylhalofantrine formed under alkali

Publisher: Elsevier BV

Date: 03-1995

DOI: 10.1016/0731-7085(95)01256-K

Abstract: The development of a new and simplified, validated LC assay for the quantitation of halofantrine and desbutylhalofantrine in plasma is described. The methodology employs an inexpensive, rapid and simple liquid-liquid extraction procedure in combination with previously reported chromatographic conditions. The method has been employed to study aspects of the pharmacokinetics of orally administered halofantrine in beagle dogs and some preliminary data are presented. During development of the extraction procedure, degradation of desbutylhalofantrine was observed under non-acidic conditions in the extraction solvent (tert-butyl methyl ether) and we also report the structural elucidation of the breakdown product and the conditions required to avoid this degradation.

A ligand-induced structural change in fatty acid– binding protein 1 is associated with potentiation of peroxisome proliferator–activated receptor agonists

Publisher: Elsevier BV

Date: 03-2019

DOI: 10.1074/JBC.RA118.006848

Activation of the pseudokinase MLKL unleashes the four-helix bundle domain to induce membrane localization and necroptotic cell death

Publisher: Proceedings of the National Academy of Sciences

Date: 06-10-2014

DOI: 10.1073/PNAS.1408987111

Abstract: The four-helix bundle (4HB) domain of Mixed Lineage Kinase Domain-Like (MLKL) bears two clusters of residues that are required for cell death by necroptosis. Mutations within a cluster centered on the α4 helix of the 4HB domain of MLKL prevented its membrane translocation, oligomerization, and ability to induce necroptosis. This cluster is composed principally of acidic residues and therefore challenges the idea that the 4HB domain engages negatively charged phospholipid membranes via a conventional positively charged interaction surface. The importance of membrane translocation to MLKL-mediated death is supported by our identification of a small molecule that binds the MLKL pseudokinase domain and retards membrane translocation to inhibit necroptotic signaling.

Role of Nox inhibitors plumbagin, ML090 and gp91ds-tat peptide on homocysteine thiolactone induced blood vessel dysfunction.

Publisher: Wiley

Date: 28-07-2015

DOI: 10.1111/1440-1681.12427

Abstract: Antioxidants have not reduced the burden of cardiovascular disease, and current evidence suggests a beneficial role of oxidative stress, via NADPH oxidase (Nox) upregulation, in endothelial function. Homocysteine thiolactone (HcyT) induces blood vessel dysfunction and this correlates with increased vascular oxidative stress. This study aimed to determine if pharmacological inhibition of Nox could impair HcyT induced blood vessel dysfunction. Abdominal aorta were excised from New Zealand White rabbits (n = 6), cut into rings and sequentially mounted in organ baths. Rings were preincubated with 0.55 μmol/L homocysteine thiolactone for 1 h, or combinations of putative Nox inhibitors (plumbagin for Nox4, gp91ds-tat for Nox2, and ML090 for Nox1), 30 min prior to the addition of HcyT, followed by a dose response curve to acetylcholine on phenylephrine preconstricted rings. Plumbagin, ML090 + gp91ds-tat and HcyT reduced responses to acetylcholine, and Plumbagin + Hcyt caused constriction to acetylcholine, which was normalised to plumbagin by ML090. Plumbagin + ML090 or plumbagin + gp91ds-tat completely impaired the effect of acetylcholine. ML090 inhibited the effect of HcyT on reduced response to acetylcholine, whereas gp91ds-tat had no effect. This study concludes that inhibition of Nox1 prevents, whereas inhibition of Nox4 worsens, acetylcholine induced blood vessel relaxation caused by HcyT, while Nox2 inhibition has no effect. However combinations of Nox inhibitors worsen acetylcholine induced blood vessel relaxation. These results suggest that there is cross-talk between Nox isoforms during physiological and pathophysiological processes.

The Role of Oxidoreductases in Determining the Function of the Neisserial Lipid A Phosphoethanolamine Transferase Required for Resistance to Polymyxin

Publisher: Public Library of Science (PLoS)

Date: 12-09-2014

DOI: 10.1371/JOURNAL.PONE.0106513

Cyclic Hexapeptide Mimics of the LEDGF Integrase Recognition Loop in Complex with HIV-1 Integrase.

Publisher: Wiley

Date: 06-07-2018

DOI: 10.1002/CMDC.201800129

Abstract: The p75 splice variant of lens epithelium-derived growth factor (LEDGF) is a 75 kDa protein, which is recruited by the human immunodeficiency virus (HIV) to tether the pre-integration complex to the host chromatin and promote integration of proviral DNA into the host genome. We designed a series of small cyclic peptides that are structural mimics of the LEDGF binding domain, which interact with integrase as potential binding inhibitors. Herein we present the X-ray crystal structures, NMR studies, SPR analysis, and conformational studies of four cyclic peptides bound to the HIV-1 integrase core domain. Although the X-ray studies show that the peptides closely mimic the LEDGF binding loop, the measured affinities of the peptides are in the low millimolar range. Computational analysis using conformational searching and free energy calculations suggest that the low affinity of the peptides is due to mismatch between the low-energy solution and bound conformations.

Binding of CFA/I pili of enterotoxigenic Escherichia coli to asialo-GM1 is mediated by the minor pilin CfaE

Publisher: American Society for Microbiology

Date: 05-2016

DOI: 10.1128/IAI.01562-15

Abstract: CFA/I pili are representatives of a large family of related pili that mediate the adherence of enterotoxigenic Escherichia coli to intestinal epithelial cells. They are assembled via the alternate chaperone-usher pathway and consist of two subunits, CfaB, which makes up the pilus shaft and a single pilus tip-associated subunit, CfaE. The current model of pilus-mediated adherence proposes that CFA/I has two distinct binding activities the CfaE subunit is responsible for binding to receptors of unknown structure on erythrocyte and intestinal epithelial cell surfaces, while CfaB binds to various glycosphingolipids, including asialo-GM1. In this report, we present two independent lines of evidence that, contrary to the existing model, CfaB does not bind to asialo-GM1 independently of CfaE. Neither purified CfaB subunits nor CfaB assembled into pili bind to asialo-GM1. Instead, we demonstrate that binding activity toward asialo-GM1 resides in CfaE and this is essential for pilus binding to Caco-2 intestinal epithelial cells. We conclude that the binding activities of CFA/I pili for asialo-GM1, erythrocytes, and intestinal cells are inseparable, require the same amino acid residues in CfaE, and therefore depend on the same or very similar binding mechanisms.

Identification of the Binding Site of Apical Membrane Antigen 1 (AMA1) Inhibitors Using a Paramagnetic Probe.

Publisher: Wiley

Date: 13-02-2019

DOI: 10.1002/CMDC.201800802

Abstract: Apical membrane antigen 1 (AMA1) is essential for the invasion of host cells by malaria parasites. Several small-molecule ligands have been shown to bind to a conserved hydrophobic cleft in Plasmodium falciparum AMA1. However, a lack of detailed structural information on the binding pose of these molecules has hindered their further optimisation as inhibitors. We have developed a spin-labelled peptide based on RON2, the native binding partner of AMA1, to probe the binding sites of compounds on PfAMA1. The crystal structure of this peptide bound to PfAMA1 shows that it binds at one end of the hydrophobic groove, leaving much of the binding site unoccupied and allowing fragment hits to bind without interference. In paramagnetic relaxation enhancement (PRE)-based NMR screening, the

Fatty Acid Binding Proteins: Potential Chaperones of Cytosolic Drug Transport in the Enterocyte?

Publisher: Springer Science and Business Media LLC

Date: 27-04-2011

DOI: 10.1007/S11095-011-0446-1

Abstract: Several poorly water-soluble drugs have previously been shown to bind to intestinal (I-FABP) and liver fatty acid binding protein (L-FABP) in vitro. The purpose of this study was to examine the potential role of drug binding to FABPs on intestinal permeability and gut wall metabolism in vivo. The intestinal permeability of ibuprofen, progesterone and midazolam (which bind FABPs) and propranolol (which does not) was examined using an autoperfused recirculating permeability model in control rats and rats where FABP levels were upregulated via pre-feeding a fat-rich diet. The intestinal permeability of drugs which bind FABPs in vitro was increased in animals where FABP levels were upregulated by prefeeding a high fat diet. The gut wall metabolism of midazolam was also reduced in animals with elevated FABP levels. Consistent with their role in the cellular transport of endogenous lipophilic substrates, FABPs appear to facilitate the intracellular disposition of drug molecules that bind FABPs in vitro. Drug binding to FABPs in the enterocyte may also attenuate gut wall metabolism in a manner analogous to the reduction in hepatic extraction mediated by drug binding to plasma proteins in the systemic circulation.

DSB proteins and bacterial pathogenicity

Publisher: Springer Science and Business Media LLC

Date: 09-02-2009

DOI: 10.1038/NRMICRO2087

Abstract: If DNA is the information of life, then proteins are the machines of life--but they must be assembled and correctly folded to function. A key step in the protein-folding pathway is the introduction of disulphide bonds between cysteine residues in a process called oxidative protein folding. Many bacteria use an oxidative protein-folding machinery to assemble proteins that are essential for cell integrity and to produce virulence factors. Although our current knowledge of this machinery stems largely from Escherichia coli K-12, this view must now be adjusted to encompass the wider range of disulphide catalytic systems present in bacteria.

Characterization of the DsbA Oxidative Folding Catalyst fromPseudomonas aeruginosaReveals a Highly Oxidizing Protein that Binds Small Molecules

Publisher: Mary Ann Liebert Inc

Date: 15-04-2010

DOI: 10.1089/ARS.2009.2736

Abstract: Bacterial antibiotic resistance is an emerging global crisis, and treatment of multidrug-resistant gram-negative infections, particularly those caused by the opportunistic human pathogen Pseudomonas aeruginosa, remains a major challenge. This problem is compounded by a lack of new antibiotics in the development pipeline: only two new classes have been developed since the 1960s, and both are indicated for multidrug-resistant gram-positive infections. A promising new approach to combat antibiotic resistance is by targeting bacterial virulence, rather than bacterial viability. The bacterial periplasmic protein DsbA represents a central point for antivirulence intervention because its oxidoreductase activity is essential for the folding and function of almost all exported virulence factors. Here we describe the three-dimensional structure of this DsbA target from P. aeruginosa, and we establish for the first time that a member of this enzyme family is capable of binding small molecules. We also describe biochemical assays that validate the redox activity of PaDsbA. Together, the structural and functional characterization of PaDsbA provides the basis for future studies aimed at designing a new class of antivirulence compounds to combat antibiotic-resistant P. aeruginosa infection.

A Backbone Linker for BOC-Based Peptide Synthesis and On-Resin Cyclization:  Synthesis of Stylostatin 1^,

Publisher: American Chemical Society (ACS)

Date: 04-1999

DOI: 10.1021/JO9818780

Abstract: We have developed a new 4-alkoxybenzyl-derived linker that anchors the C-terminal amino acid to the resin through the alpha-nitrogen atom. The linker allows BOC solid-phase peptide assembly and peptide cleavage using standard HF protocols. This linking strategy provides a versatile on-resin route to cyclic peptides and avoids the diketopiperazine formation that is prominent when using FMOC chemistry on backbone linkers. The linker was prepared by forming the aryl ether from 4-hydroxybenzaldehyde and bromovaleric acid. Subsequent reductive amination of the aldehyde with an allyl-protected amino acid ester and acylation of the resulting secondary amine provided the tertiary amide. After linking the amide to the resin, standard BOC SPPS, followed by allyl deprotection, cyclization, and HF cleavage gave cyclic peptides in high purity. To exemplify the strategy, the cytotoxic heptapeptide, stylostatin 1, was synthesized from two linear precursors. For comparison purposes, the yields of the on-resin and solution-phase cyclization were determined and found to be dependent upon the linear precursor. This linker technology provides new solid-phase avenues in accessing libraries of cyclic peptides.

The Fragment-Based Development of a Benzofuran Hit as a New Class of Escherichia coli DsbA Inhibitors

Publisher: MDPI AG

Date: 18-10-2019

DOI: 10.3390/MOLECULES24203756

Abstract: A fragment-based drug discovery approach was taken to target the thiol-disulfide oxidoreductase enzyme DsbA from Escherichia coli (EcDsbA). This enzyme is critical for the correct folding of virulence factors in many pathogenic Gram-negative bacteria, and small molecule inhibitors can potentially be developed as anti-virulence compounds. Biophysical screening of a library of fragments identified several classes of fragments with affinity to EcDsbA. One hit with high mM affinity, 2-(6-bromobenzofuran-3-yl)acetic acid (6), was chemically elaborated at several positions around the scaffold. X-ray crystal structures of the elaborated analogues showed binding in the hydrophobic binding groove adjacent to the catalytic disulfide bond of EcDsbA. Binding affinity was calculated based on NMR studies and compounds 25 and 28 were identified as the highest affinity binders with dissociation constants (KD) of 326 ± 25 and 341 ± 57 µM respectively. This work suggests the potential to develop benzofuran fragments into a novel class of EcDsbA inhibitors.

Conformationally Constrained Macrocycles That Mimic Tripeptide β-Strands in Water and Aprotic Solvents

Publisher: American Chemical Society (ACS)

Date: 24-04-2002

DOI: 10.1021/JA0256461

Abstract: The beta-strand conformation is unknown for short peptides in aqueous solution, yet it is a fundamental building block in proteins and the crucial recognition motif for proteolytic enzymes that enable formation and turnover of all proteins. To create a generalized scaffold as a peptidomimetic that is pre-organized in a beta-strand, we in idually synthesized a series of 15-22-membered macrocyclic analogues of tripeptides and analyzed their structures. Each cycle is highly constrained by two trans amide bonds and a planar aromatic ring with a short nonpeptidic linker between them. A measure of this ring strain is the restricted rotation of the component tyrosinyl aromatic ring (DeltaG(rot) 76.7 kJ mol(-1) (16-membered ring), 46.1 kJ mol(-1) (17-membered ring)) evidenced by variable temperature proton NMR spectra (DMF-d(7), 200-400 K). Unusually large amide coupling constants ((3)J(NH-CHalpha) 9-10 Hz) corresponding to large dihedral angles were detected in both protic and aprotic solvents for these macrocycles, consistent with a high degree of structure in solution. The temperature dependence of all amide NH chemical shifts (Deltadelta/T 7-12 ppb/deg) precluded the presence of transannular hydrogen bonds that define alternative turn structures. Whereas similar sized conventional cyclic peptides usually exist in solution as an equilibrium mixture of multiple conformers, these macrocycles adopt a well-defined beta-strand structure even in water as revealed by 2-D NMR spectral data and by a structure calculation for the smallest (15-membered) and most constrained macrocycle. Macrocycles that are sufficiently constrained to exclusively adopt a beta-strand-mimicking structure in water may be useful pre-organized and generic templates for the design of compounds that interfere with beta-strand recognition in biology.

Fragment screening libraries for the identification of protein hot spots and their minimal binding pharmacophores

Publisher: Royal Society of Chemistry (RSC)

Date: 2023

DOI: 10.1039/D2MD00253A

Abstract: Small molecule interaction hotpots were identified by screening small, low complexity fragments using X-ray crystallography. These hot spots include cryptic pockets and provide pharmacophore mapping that may be used in structure-based drug design.

Preparation, crystallization and preliminary X-ray diffraction analysis of two intestinal fatty-acid binding proteins in the presence of 11-(dansylamino)undecanoic acid

Publisher: International Union of Crystallography (IUCr)

Date: 27-01-2011

DOI: 10.1107/S1744309110051481

Assignments of human integrin α1I domain in the apo and Mg ²⁺ bound states

Publisher: Springer Science and Business Media LLC

Date: 22-01-2014

DOI: 10.1007/S12104-013-9465-7

Abstract: The α1β1 integrin receptor binds to its main extracellular ligand, collagen, through an inserted domain in its α-subunit called the αI domain (αI). αI contains a metal binding site that allows collagen to coordinate to the domain through a alent metal ion. Here we report the backbone assignments of the apo and Mg(2+) bound state of the isolated human α1I and the chemical shift changes resulting from metal coordination.

Solution structure of the cardiostimulant polypeptide anthopleurin-B and comparison with anthopleurin-A

Publisher: Elsevier BV

Date: 08-1995

DOI: 10.1016/S0969-2126(01)00214-3

Abstract: The polypeptide anthopleurin-B (AP-B) is one of a number of related toxins produced by sea anemones. AP-B delays inactivation of the voltage-gated sodium channel of excitable tissue. In the mammalian heart, this effect is manifest as an increase in the force of contraction. As a result, there is interest in exploiting the anthopleurins as lead compounds in the design of novel cardiac stimulants. Essential to this endeavour is a high-resolution solution structure of the molecule describing the positions of functionally important side chains. AP-B exists in multiple conformations in solution as a result of cis-trans isomerization about the Gly40-Pro41 peptide bond. The solution structure of the major conformer of AP-B has been determined by two-dimensional 1H NMR at pH 4.5 and 25 degrees C. The core structure is a four-stranded, antiparallel beta-sheet (residues 2-4, 20-23, 34-37 and 45-48) and includes several beta-turns (6-9, 25-28, 30-33). Three loops connect the beta-strands, the longest and least well defined being the first loop, extending from residues 8-17. These features are shared by other members of this family of sea anemone toxins. The locations of a number of side chains which are important for the cardiac stimulatory activity of AP-B are well defined in the structures. We have described the solution structure of AP-B and compared it with that of AP-A, from which it differs by substitutions at seven amino acid positions. It shares an essentially identical fold with AP-A yet is about 10-fold more active. Comparison of the structures, particularly in the region of residues essential for activity, gives a clearer indication of the location and extent of the cardioactive pharmacophore in these polypeptides.

Identification and characterization of two drug-like fragments that bind to the same cryptic binding pocket of Burkholderia pseudomallei DsbA

Publisher: International Union of Crystallography (IUCr)

Date: 2022

DOI: 10.1107/S2059798321011475

Abstract: Disulfide-bond-forming proteins (Dsbs) play a crucial role in the pathogenicity of many Gram-negative bacteria. Disulfide-bond-forming protein A (DsbA) catalyzes the formation of the disulfide bonds necessary for the activity and stability of multiple substrate proteins, including many virulence factors. Hence, DsbA is an attractive target for the development of new drugs to combat bacterial infections. Here, two fragments, bromophenoxy propanamide ( 1 ) and 4-methoxy- N -phenylbenzenesulfonamide ( 2 ), were identified that bind to DsbA from the pathogenic bacterium Burkholderia pseudomallei , the causative agent of melioidosis. The crystal structures of oxidized B. pseudomallei DsbA (termed BpsDsbA) co-crystallized with 1 or 2 show that both fragments bind to a hydrophobic pocket that is formed by a change in the side-chain orientation of Tyr110. This conformational change opens a `cryptic' pocket that is not evident in the apoprotein structure. This binding location was supported by 2D-NMR studies, which identified a chemical shift perturbation of the Tyr110 backbone amide resonance of more than 0.05 p.p.m. upon the addition of 2 m M fragment 1 and of more than 0.04 p.p.m. upon the addition of 1 m M fragment 2 . Although binding was detected by both X-ray crystallography and NMR, the binding affinity ( K d ) for both fragments was low (above 2 m M ), suggesting weak interactions with BpsDsbA. This conclusion is also supported by the crystal structure models, which ascribe partial occupancy to the ligands in the cryptic binding pocket. Small fragments such as 1 and 2 are not expected to have a high energetic binding affinity due to their relatively small surface area and the few functional groups that are available for intermolecular interactions. However, their simplicity makes them ideal for functionalization and optimization. The identification of the binding sites of 1 and 2 to BpsDsbA could provide a starting point for the development of more potent novel antimicrobial compounds that target DsbA and bacterial virulence.

Backbone and side chain 1H, 15N and 13C assignments for the reduced form of the oxidoreductase protein DsbA from Vibrio cholerae

Publisher: Springer Science and Business Media LLC

Date: 05-06-2007

DOI: 10.1007/S12104-007-9018-Z

Abstract: We have determined 13C/15N/1H assignments for the reduced and oxidised forms of Vibrio cholerae DsbA (VcDsbA). These form the basis for ongoing studies aimed at characterising the dynamics observed in the different redox forms of this bacterial oxidoreductase enzyme.

Production, biophysical characterization and initial crystallization studies of the N- and C-terminal domains of DsbD, an essential enzyme inNeisseria meningitidis

Publisher: International Union of Crystallography (IUCr)

Date: 2018

DOI: 10.1107/S2053230X17017800

Abstract: The membrane protein DsbD is a reductase that acts as an electron hub, translocating reducing equivalents from cytoplasmic thioredoxin to a number of periplasmic substrates involved in oxidative protein folding, cytochrome c maturation and oxidative stress defence. DsbD is a multi-domain protein consisting of a transmembrane domain (t-DsbD) flanked by two periplasmic domains (n-DsbD and c-DsbD). Previous studies have shown that DsbD is required for the survival of the obligate human pathogen Neisseria meningitidis . To help understand the structural and functional aspects of N. meningitidis DsbD, the two periplasmic domains which are required for electron transfer are being studied. Here, the expression, purification and biophysical properties of n- Nm DsbD and c- Nm DsbD are described. The crystallization and crystallographic analysis of n- Nm DsbD and c- Nm DsbD are also described in both redox states, which differ only in the presence or absence of a disulfide bond but which crystallized in completely different conditions. Crystals of n- Nm DsbD Ox , n- Nm DsbD Red , c- Nm DsbD Ox and c- Nm DsbD Red diffracted to 2.3, 1.6, 2.3 and 1.7 Å resolution and belonged to space groups P 2 1 3, P 321, P 4 1 and P 12 1 1, respectively.

Shanghai Jiao Tong University

Location: China

View Organisation

Shanghai Jiao Tong University School Of Materials Science And Engineering

Location: China

View Organisation

City College Of Science & Commerce

Location: Pakistan

View Organisation

Govt. College For Women

Location: Pakistan

View Organisation

Mamoona Postgraduate College For Women

Location: Pakistan

View Organisation

NFC Institute Of Engineering & Technology

Location: Pakistan

View Organisation

Biomolecular Research Institute

Location: Australia

View Organisation

Monash University

Location: Australia

View Organisation

Bahauddin Zakariya University

Location: Pakistan

View Organisation

Monash Institute Of Pharmaceutical Sciences

Location: Australia

View Organisation

University Of Nottingham

Location: United Kingdom of Great Britain and Northern Ireland

View Organisation

University Of Leeds

Location: United Kingdom of Great Britain and Northern Ireland

View Organisation

Anti-Malarial Agents Targeting Apical Membrane Antigen 1

Start Date: 2016

End Date: 2018

Funder: National Health and Medical Research Council

Melbourne Biomolecular NMR Network

Start Date: 2009

End Date: 2009

Funder: Australian Research Council

Cellular Pathways Of Nuclear Receptor Activation

Start Date: 2015

End Date: 2019

Funder: Australian Research Council

Distributed Facility For Fragment Based Drug Discovery

Start Date: 2016

End Date: 2016

Funder: Australian Research Council

DsbA: A Target For The Design Of Drug Candidates As Selective Inhibitors Of Oxidative Protein Folding In Gram Negative Bacteria

Start Date: 2004

End Date: 2006

Funder: Australian Research Council

Dissemination And Virulence Properties Of The She Pathogenicity Island Of Shigella Flexneri.

Start Date: 2003

End Date: 2012

Funder: National Health and Medical Research Council

Targeting Virulence Of Pseudomonas Aeruginosa By Inhibiting Oxidative Protein Folding

Start Date: 2009

End Date: 2012

Funder: Australian Research Council

Drug Binding To Human Fatty Acid Binding Proteins: A Mechanism Of Cellular Transport For Poorly Water Soluble Drugs

Start Date: 2006

End Date: 2008

Funder: Australian Research Council

Structure-based Design Of Novel Therapeutics For Multi-drug Resistant Neisseria Gonorrhoeae

Start Date: 2015

End Date: 2017

Funder: National Health and Medical Research Council

Acoustic Liquid Handling Robotics For Bioactive Compound Discovery

Start Date: 2017

End Date: 2017

Funder: Australian Research Council

The Role Of Fatty Acid Binding Proteins In The Binding And Transport Of Lipophilic Drugs

Start Date: 2003

End Date: 2005

Funder: Australian Research Council

Small Molecule NMR Facility For Accelerated Drug Discovery

Start Date: 2004

End Date: 2004

Funder: Australian Research Council

Guiding Ligands To Nuclear Receptors: The Role Of Fatty Acid Binding Proteins

Start Date: 2009

End Date: 2011

Funder: Australian Research Council

A 700 MHz Nuclear Magnetic Resonance (NMR) Spectrometer For The Melbourne Biomolecular NMR Network: A High Throughput Resource

Start Date: 2012

End Date: 2012

Funder: Australian Research Council

ARC Training Centre For The Development Of Tools For Fragment Based Design

Start Date: 2018

End Date: 2022

Funder: Australian Research Council

DsbA Inhibitors: From Hits To Leads

Start Date: 2016

End Date: 2018

Funder: National Health and Medical Research Council

New Approaches To Inhibition Of Activity Of HIV Integrase

Start Date: 2010

End Date: 2012

Funder: Australian Research Council

Therapeutic Approaches To Treat Human Immunodeficiency Virus Infection: Development Of HIV-1 Integrase Inhibitors

Start Date: 2007

End Date: 2009

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0664069

Start Date: 01-2006

End Date: 12-2008

Amount: $276,000.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP0455508

Start Date: 2004

End Date: 01-2007

Amount: $103,905.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0342458

Start Date: 2003

End Date: 06-2006

Amount: $255,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP200100796

Start Date: 2020

End Date: 12-2022

Amount: $670,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP150102587

Start Date: 2015

End Date: 12-2019

Amount: $563,000.00

Funder: Australian Research Council

Industrial Transformation Training Centres - Grant ID: IC180100021

Start Date: 07-2019

End Date: 08-2025

Amount: $4,163,359.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0987500

Start Date: 2009

End Date: 06-2012

Amount: $360,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE210100130

Start Date: 05-2021

End Date: 12-2022

Amount: $1,000,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0989504

Start Date: 07-2009

End Date: 07-2010

Amount: $1,400,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0453651

Start Date: 12-2003

End Date: 12-2006

Amount: $907,511.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE120100022

Start Date: 02-2012

End Date: 12-2012

Amount: $480,000.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP0455345

Start Date: 09-2004

End Date: 02-2008

Amount: $70,668.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP130100740

Start Date: 03-2014

End Date: 12-2016

Amount: $590,625.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP100100194

Start Date: 03-2011

End Date: 06-2013

Amount: $600,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE160100047

Start Date: 2016

End Date: 09-2016

Amount: $380,000.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP200100540

Start Date: 07-2020

End Date: 12-2023

Amount: $433,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE170100075

Start Date: 2017

End Date: 12-2017

Amount: $315,000.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP0990166

Start Date: 03-2010

End Date: 12-2012

Amount: $600,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE140100162

Start Date: 2014

End Date: 2015

Amount: $300,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP120102930

Start Date: 2012

End Date: 08-2015

Amount: $410,000.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP0775192

Start Date: 01-2007

End Date: 12-2009

Amount: $377,513.00

Funder: Australian Research Council